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.

Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown. Leptospirosis is an important but largely under-recognized public health problem in the tropics. It is a re-emerging disease and outbreaks of leptospirosis have been frequently reported in Sri Lanka. Most of the cases, however, were clinically diagnosed, and Leptospira serotypes and genotypes currently prevailing in the country remain unknown. For the prevention of leptospirosis, doxycycline prophylaxis and health education (early medical help, use of protective wear, etc) are implemented in farmers, which is the highest risk group, in endemic areas. In the present study, we revealed that genetically diverse Leptospira strains presently cause human leptospirosis in this country. Novel STs were isolated during an outbreak in Southern Province and may be related to the recent surge in leptospirosis patients with new clinical entities. The emergence of new STs also implies the importance of strict quarantine for imported domestic animals that are potential reservoirs of Leptospira spp. as well as the unintentional introduction of rodent reservoirs and the existence of unidentified reservoir animals in this country. New information from this study will be valuable in future research for patient management and specific targeted control approaches for reservoir hosts in the prevention and control of leptospirosis in Sri Lanka.

Hantaan virus is the causative agent of hemorrhagic fever with renal syndrome (HFRS). The Hantaan virus strain, Korean hemorrhagic fever virus clone-5 (KHF5), causes weight loss and renal hemorrhage in laboratory mice. Clone-4 (KHF4), which has a single E417K amino acid change in its glycoprotein, is an avirulent variant. In this study, KHF4 and KHF5 were compared to evaluate pathological differences in mice in vitro and in vivo. The characteristics of the two glycoproteins were not significantly different in vitro. However, the virulent KHF5 strain targeted the lungs and caused pneumonia and edema in vivo. Both strains induced high infectivity levels in the liver and caused hepatitis; however, petechial hemorrhage and glycogen storage reduction were observed in KHF5-infected mice alone. Renal hemorrhage was observed using viral antigens in the tubular region of KHF5-infected mice. In addition, an increase in white blood cell levels and neutrophilia were found in KHF5-infected mice. Microarray analysis of liver cells showed that CD8+ T cell activation, acute-phase protein production, and neutrophil activation was induced by KHF5 infection. KHF5 infectivity was significantly increased in vivo and the histological and clinicopathological findings were similar to those in patients with HFRS.

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.

Wild rodents are natural hosts of Leptospira spp. and are exposed to various pesticides, some of which are immunotoxic. Rodent urine is an important source of infection for humans and other animals. We evaluated the effects of pesticide exposure on Leptospira growth in mice. Diazinon, at doses of 0.2, 1, and 5 mg/kg/day, was orally administered continuously to mice infected with Leptospira interrogans serogroup Hebdomadis for 32 days. The numbers of L. interrogans in urine and kidney tissues were significantly lower in mice exposed to 5 mg/kg/day diazinon than in unexposed mice (p < 0.05). The urinary concentration of 2-isopropyl-6-methyl-4-pyrimidinol, the metabolite of diazinon, was comparable with the concentration at which viability of L. interrogans was decreased in in vitro assay, suggesting that it had toxic effects on L. interrogans in the proximal renal tubules. Diazinon exposure reinforced Leptospira-induced expression of inflammatory cytokine genes in kidney tissues, and an enhanced immune system might suppress Leptospira growth. These results suggest that diazinon exposure may not increase the risk of Leptospira transmission to humans through mice. This novel study evaluated the relationship between pesticide exposure and Leptospira infection in mice, and the results could be useful for risk assessment of leptospirosis.

Objective: Chronic kidney disease (CKD) and chronic kidney disease of uncertain etiology (CKDu) are chronic kidney diseases that pose a significant health burden in Sri Lanka. Leptospirosis is a bacterial zoonosis that primarily damages renal tissues by colonization of Leptospira spp. in the renal tubules and is a suspected etiological agent of CKDu. Since Sri Lanka is an endemic for leptospirosis and outbreaks of the disease have been reported, this study aimed to determine the association between leptospirosis and chronic kidney disease in two geographically distinct regions of Sri Lanka, Badulla (CKDu endemic) and Kandy (CKDu non-endemic) districts. Results: Eighty-five patients with CKDu and 149 controls from Badulla and 49 patients with CKD and 135 controls from Kandy were serologically tested by microscopic agglutination test with a panel of 11 Leptospira serogroups. The seroprevalence rates for leptospirosis were 7.1% and 13.4% in the CKDu and control groups, respectively, in Badulla and 2.1% and 18.5% in the CKD and control groups, respectively, in Kandy. There were no statistically significant differences between demographic characteristics and leptospirosis seropositivity in the CKD and control groups in either Badulla or Kandy.