Identification of cap-dependent endonuclease inhibitors with broad-spectrum activity against bunyaviruses.

Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.

Hantavirus infection as a risk factor for chronic kidney disease of unknown aetiology (CKDu) and its prevalence in endemic areas of Sri Lanka since 2010 according to a retrospective serological analysis.

Background. Chronic kidney disease of unknown aetiology (CKDu) is a major public health problem in Sri Lanka, especially among agrarian communities. Although the cause of CKDu is still unknown, hantavirus infection has been proposed as a risk factor.Methods. This study was performed using serological samples collected from two CKDu-endemic areas, Anuradhapura (2010) and Badulla districts (2010 and 2016), and a non-endemic area, Matale (2016) district. The presence of anti-Thailand orthohantavirus IgG antibodies was investigated in serum samples. Hantavirus seroprevalence and demographic data were epidemiologically analysed.Results. Seroprevalence was higher in CKDu patients (40.6-60.0 %) and healthy individuals in CKDu-endemic areas (17.6-25.5 %) than in healthy individuals in non-endemic areas (3.0 %). Statistically significant odds ratios (ORs) for hantavirus infection in CKDu patients were detected in CKDu-endemic areas [ORs: 3.2 and 3.1; 95 % confidence interval (CI): 1.8-5.5 and 1.8-5.2 in Anuradhapura and Badulla districts in 2010; and OR: 4.4, 95 % CI: 2.3-8.5 in 2016 in Badulla district). Furthermore, the OR for hantavirus infection in Badulla district has increased in the last decade from 3.1 (95 % CI: 1.8-5.3) to 4.4 (95 % CI: 2.3-8.5).Conclusion. Hantavirus infection has been prevalent in two distant CKDu-endemic areas since 2010. The observed significant association of hantavirus seropositivity with CKDu indicates a possible role of hantavirus infection in CKDu pathogenesis.

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.

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.

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.

Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay.

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.

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.

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 [...].

Multiplex Immunochromatographic Assay for Serologic Diagnosis of Major Infectious Diseases in Laboratory Mice.

Serologic monitoring of infectious diseases is important for microbial control in colonies of laboratory mice. Rapid and simple tests that do not require killing animals are valuable for this purpose. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to mouse hepatitis virus (MHV), Sendai virus (also known as hemagglutinating virus of Japan [HVJ]), and Clostridium piliforme (The pathogen that causes Tyzzer disease), which are major infectious diseases in mice. For this assay, an ICA strip was put into a microtube containing 150 µL PBS and either 0.75 µL mouse serum or 1.5 µL whole blood. Binding antibodies were visualized by using protein A-conjugated colloidal gold. Under these conditions, multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. To evaluate the sensitivity and specificity of multiplex ICA, positive serum samples for each infectious disease were used. Sensitivities of the multiplex ICA test for MHV, HVJ, and C. piliforme were 100%, 100%, and 90%, respectively. No nonspecific reaction was observed in any of the 30 positive sera. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA test. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid, simple, and safe serologic testing of laboratory mice.

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.

Comparison of immune response in mice sensitized to an animal allergen, Can f 1, and to a food allergen, ovalbumin.

Can f 1 belongs to the lipocalin superfamily and is considered to be an animal allergen. The immune response induced by Can f 1 in mice was compared with that induced by ovalbumin (OVA), a typical food allergen. Female BALB/c and C57BL/6 mice (6 weeks of age) were subcutaneously injected with Can f 1 or OVA with or without aluminum hydroxide (Alum) three times with intervals of two weeks. Serum levels of total IgE or antigen-specific IgE and production of IL13 and IFNγ from splenocytes were analyzed. Immunization with Can f 1 or OVA increased serum levels of both total IgE and antigen-specific IgE significantly irrespective of Alum. These results indicate that Can f 1 and OVA were able to induce allergic sensitization in mice. Splenocyte production of IL13 in mice immunized with Can f 1 or OVA with and without Alum were significantly increased after stimulation with each antigen. However, IL13 levels in the mice immunized with Can f 1 with Alum were significantly lower than those immunized without Alum. Increases in IFNγ levels after stimulation with Can f 1 or OVA were not remarkable. No influence of genetic backgrounds of BALB/c and C57BL/6 mice was found. Although Can f 1 induced Th2 type immune responses as was also the case for immunization with OVA, an inhibitory effect of Alum on induction of IL13 was observed only in mice immunized with Can f 1. These results suggest that the immune mechanism for allergic sensitization with Can f 1 is different from that with OVA.

Development and evaluation of monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for the diagnosis of acute leptospirosis in humans.

Monoclonal antibody (MAb)-based ELISA was developed to detect leptospiral antigens from the plasma of febrile patients. The MAb reacts specifically with pathogenic leptospires and the assay possesses excellent diagnostic test parameters compared to PCR, indicating that this new ELISA is useful for the early diagnosis of leptospirosis with low operational cost.

Expression of a Recombinant Nucleocapsid Protein of Rift Valley Fever Virus in Vero Cells as an Immunofluorescence Antigen and Its Use for Serosurveillance in Traditional Cattle Herds in Zambia.

The open reading frame of the nucleocapsid protein (NP) of Rift Valley fever virus (RVFV) strain MP12 was cloned and expressed in Vero E6 cells. The recombinant NP (rNP)-expressing cells were used as antigens for an indirect immunofluorescent antibody assay (IFA). The rNP-based IFA and RVFV-infected Vero E6 cell (authentic antigen)-based IFA showed similar IFA profiles with immune rabbit serum, which was prepared by immunization with rNP expressed using a baculovirus vector. A total of 942 traditional cattle sera obtained in five districts in Central, Southern, and Western provinces of Zambia were screened for anti-RVFV antibodies by the authentic antigen-based and rNP-based IFAs. Significant agreement was obtained between the two IFAs. The findings show that the rNP-based IFA is a safe and useful diagnostic tool as an alternative to the authentic antigen-based IFA. The antibody titers given by the rNP-based IFA were higher than those by the authentic antigen-based IFA. Therefore, the rNP-based IFA might be useful for serosurveillance of RVFV infection among cattle. Antibody prevalence rates in the five districts were 1.3% to 13.5% in the authentic antigen-based IFA and 6.0% to 21.4% in the rNP-based IFA. The results indicated that despite no reports of active cases of RVF in these provinces of Zambia, the virus is circulating among cattle herds.

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.

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.