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Adaptation of the viral life cycle to host cells is necessary for efficient viral infection and replication. This evolutionary process has contributed to the mechanism for determining the host range of viruses. Orthobornaviruses, members of the family Bornaviridae, are non-segmented, negative-strand RNA viruses, and several genotypes have been isolated from different vertebrate species. Previous studies revealed that some genotypes isolated from avian species can replicate in mammalian cell lines, suggesting the zoonotic potential of avian orthobornaviruses. However, the mechanism by which the host specificity of orthobornaviruses is determined has not yet been identified. In this study, we found that the infectivity of orthobornaviruses is not determined at the viral entry step, mediated by the viral glycoprotein and matrix protein. Furthermore, we demonstrated that the nuclear localization signal (NLS) sequence in the viral nucleoprotein (N) has evolved under natural selection and determines the host-specific viral polymerase activity. A chimeric mammalian orthobornavirus, which has the NLS sequence of avian orthobornavirus N, exhibited a reduced propagation efficiency in mammalian cells. Our findings indicated that nuclear transport of the viral N is a determinant of the host range of orthobornaviruses, providing insights into the evolution and host adaptation of orthobornaviruses.

When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, the RDV method) to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples. First, the influenza virus was inoculated into mice to prepare infected tissue samples. RNA extracted from the mouse lung homogenates was transcribed into cDNA and then analyzed using the RDV method and next-generation sequencing, using MiSeq and MinION sequencers. The RDV method was unable to detect the influenza virus in the infected tissue samples. However, influenza virus reads were detected using next-generation sequencing. Comparing MiSeq and MinION, the time required for library and sequence preparation was shorter for MinION sequencing than for MiSeq sequencing. We conclude that when a causative virus needs to be rapidly identified from an infectious sample, MinION sequencing is currently the method of choice.