Maintenance of Hokkaido virus, a genotype of <i>Orthohantavirus puumalaense</i> , in the rodent host <i>Myodes rufocanus bedfordiae</i> under natural conditions

ABSTRACT A variety of orthohantaviruses (family Hantaviridae ) have preferred natural host species, with transmission among hosts generally thought to occur through direct physical contact and inhalation of virus-contaminated excreta, although the infection to the other species occasionally occurs. Despite extensive experimental studies, the mechanisms of orthohantavirus maintenance and transmission under natural conditions remain unclear. In this study, field surveys were conducted in a forest in Tobetsu, Hokkaido, Japan, between 2022 and 2025 to capture gray red-backed voles ( Myodes rufocanus bedfordiae ), the natural host of Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense . Among 199 captured rodents, 23 were positive for HOKV infection. Five individuals were positive for viral RNA but negative for anti-HOKV IgG antibodies on ELISA and IFA and exhibited low neutralizing antibody titers and low IgG avidity indexes (≤26%), suggesting acute infection. In contrast, 18 individuals were positive for viral RNA and showed high antibody titers on ELISA, IFA, and neutralization tests, as well as high IgG avidities (≥64%); these individuals were considered persistently infected. High levels of viral RNA and antigens were consistently detected in the lungs, kidneys, and spleen during both potential acute and persistent phases of HOKV infection by quantitative PCR and immunohistochemistry. Infectious HOKV was also recovered from oral swabs (8/8), urine (3/6), and feces (4/6) of individual rodents captured in 2024. These findings showed that HOKV can persist at high viral loads in host organs and be excreted throughout the course of infection, contributing to the long-term maintenance of orthohantavirus in natural host populations. IMPORTANCE Diseases caused by zoonotic agents are major public health concerns. Orthohantaviruses are typical examples of zoonotic viruses transmitted from wild rodent hosts. Despite extensive laboratory investigations, the mechanisms of viral persistence and transmission in natural hosts remain poorly understood. Our study demonstrated that Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense , seems to establish both acute and persistent infections in wild grey red-backed voles ( Myodes rufocanus bedfordiae ) without inducing major pathological changes. Detection of infectious virus in saliva, urine, and feces revealed multiple virus-shedding routes. Saliva likely serves as the predominant source of transmission, given that infectious virus was recovered from oral swabs of all infected rodents captured in 2024. These findings expand understanding of orthohantavirus ecology, persistence, and maintenance in reservoir populations. This information is crucial for evaluating spillover risks and enhancing public health preparedness.