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.

Establishment of Subclones of the Severe Fever with Thrombocytopenia Syndrome Virus YG1 Strain Selected Using Low pH-Dependent Cell Fusion Activity.

The first clinical case of the YG1 strain of the severe fever with thrombocytopenia syndrome virus (SFTSV) has been isolated in Japan. We found that only some of the cells underwent low pH-dependent cell fusion, although all of the cells were confirmed to have been infected with the virus. This suggested that the YG1 strain consists of a heterogeneous mixture of related viruses. Here, we established 3 subclones (termed E3, A4, and B7) from the YG1 strain, using the limiting dilution method with the pH-dependent cell fusion activity. Subclone E3 showed weak fusion activity and cytopathic effects (CPE) in Vero E6 cells. The amino acid sequence of E3 was identical to the published sequence for the YG1 strain, and it likely comprises a subpopulation of the YG1 strain. Subclone A4 displayed strong fusion activity under acidic conditions. In contrast, subclone B7 showed strong fusion activity and CPE under neutral and acidic conditions. Two amino acid differences shared between B7 and A4 were found in the envelope glycoproteins. In addition, an amino acid variant of the RNA-dependent RNA polymerase was found only in B7. These subclones will be valuable tools to elucidate cell fusion mechanisms of SFTSV and the relationship between viral proteins and their functions.

Distinct genetic characteristics of Sri Lankan Rattus and Bandicota (Murinae, Rodentia) inferred from mitochondrial and nuclear markers.

We examined genetic variation in black rats (the Rattus rattus complex) from Kandy District, Sri Lanka using mitochondrial cytochrome b (cytb, 1140 bp) and nuclear melanocortin 1 receptor (Mc1r, 954 bp) gene sequences together with database sequences. We confirmed the existence of two divergent mitochondrial lineages in Sri Lankan black rats, with genetic distance of 2.2% and estimated divergence time of 0.3 million years ago. Because one lineage is unique to the island and the other is closely related to R. rattus populations on the Indian subcontinent, two migration events of R. rattus from the subcontinent are inferred, one ancient and one recent. Mc1r analyses revealed 12 haplotypes among the Sri Lankan black rats. A median-joining network together with other available sequences separated the 12 haplotypes into two groups, one unique to the island and the other related to previously reported R. rattus sequences. Notably, most individuals possessed various combinations of both haplotype groups which had no association with the cytb clades. These results imply that old and new R. rattus lineages are now intermingled as a result of hybridization in Sri Lanka. Specimens of the lesser bandicoot rat (Bandicota bengalensis) collected from Sri Lanka (n = 24) were shown to have no genetic variability in the cytb sequence. Our results indicate that the two most abundant groups of commensal rats in Sri Lanka, black rats and lesser bandicoot rats, are the product of contrasting evolutionary histories on different timescales.

Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses.

BACKGROUND: Hantaviruses are causative agents of hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. There is a need for time-saving diagnostic methods. In the present study, recombinant N antigens were used as antigens in an immunochromatography strip (ICG) test to detect specific IgG antibodies. METHODS: The N-terminal 103 amino acids (aa) of Hantaan virus (HTNV), Puumala virus (PUUV) and Andes virus (ANDV) nucleocapsid (N) protein were expressed in E. coli as representative antigens of three groups (HFRS, NE and HPS-causing viruses) of hantavirus. Five different types of ICG test strips, one antigen line on one strip for each of the three selected hantaviruses (HTNV, PUUV and ANDV), three antigen lines on one strip and a mixed antigen line on one strip, were developed and sensitivities were compared. RESULTS: A total of 87 convalescent-phase patient sera, including sera from 35 HFRS patients, 36 NE patients and 16 HPS patients, and 25 sera from healthy seronegative people as negative controls were used to evaluate the ICG test. Sensitivities of the three-line strip and mixed-line strip were similar to those of the single antigen strip (97.2 to 100%). On the other hand, all of the ICG test strips showed high specificities to healthy donors. CONCLUSION: These results indicated that the ICG test with the three representative antigens is an effective serodiagnostic tool for screening and typing of hantavirus infection in humans.

Neutrophil depletion suppresses pulmonary vascular hyperpermeability and occurrence of pulmonary edema caused by hantavirus infection in C.B-17 SCID mice.

UNLABELLED: Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice. IMPORTANCE: Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection.

Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats.

Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats.

Serological evidence of Thailand virus-related hantavirus infection among suspected leptospirosis patients in Kandy, Sri Lanka.

A cross-sectional study was undertaken to determine the current prevalence of leptospirosis and hantaviral infections, and the socio-demographic characteristics and risk factors of infected patients, in Kandy, Sri Lanka. This report discusses the serological evidence of hantavirus infections among 105 suspected leptospirosis patients, 8 of whom had hantavirus antibodies. Serotyping ELISA showed that these 8 patients had high optical density values for Thailand virus. Most of the sera showed that the focus reduction neutralization test titer against Thailand virus was higher than that against Seoul virus, thereby suggesting that the hantaviral antibodies found in Sri Lanka are different from Seoul virus but closely related to Thailand virus. These findings imply that the hantaviral infection found in Kandy, Sri Lanka appears to be due to a virus similar to Thailand virus. Epidemiological analysis revealed that the association between hantavirus infection and socio-demographic characteristics was not statistically significant.