D uring the past 20 years, coronaviruses belonging to the genus Betacoronavirus have caused multiple human epidemic or pandemic diseases, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease (COVID-19). Two viruses of the subgenus Sarbecovirus are severe acute respiratory syndrome coronavirus (SARS-CoV), which causes SARS, and SARS-CoV-2, which causes COVID-19. Although Rhinolophus spp. bats in Asia, Europe, and Africa are considered natural reservoirs of sarbecoviruses (1-3), the epidemiology and distribution of these viruses remain largely unknown, especially outside China. Previously, partial RNA-dependent RNA polymerase (RdRp) genes of betacoronaviruses were detected in little Japanese horseshoe bats (Rhinolophus cornutus) (4). However, limited sequence information left the genetic and virological properties unclear. We detected and determined the entire genome sequence of a bat sarbecovirus belonging to a phylogenetic clade that includes SARS-CoV-2 from R. cornutus bats in Japan. Further, we used a pseudotyped virus system to characterize an entry step of this virus into cells. The Study R. cornutus is an endemic bat species in Japan and is found nationwide. These bats primarily inhabit caves and abandoned tunnels in the countryside during daytime and capture insects at night outside their roosts. R. cornutus bats often cohabit with other insectivorous bats, such as R. ferrumequinum or Myotis macrodactylus, and occasionally with wild animals, such as the masked palm civet (Paguma larvata), in their daytime roosts. In 2013, we captured 4 R. cornutus bats in a cave in Iwate prefecture, Japan, and extracted RNA from fresh feces. Then, we used real-time reverse transcription PCR (rRT-PCR) to detect the partial RdRp gene of sarbecovirus from 2 samples by using a pair of primers designed to detect betacoronavirus. In 2020, we performed RNA sequencing and determined the full genome sequence of 1 sample, Rc-o319, which exhibited lower cycle threshold value by rRT-PCR. We performed a BLAST (https://blast.ncbi.nlm. nih.gov/Blast.cgi) analysis of the full genome of Rc-o319, which showed Rc-o319 had the highest nucleotide homology to SARS-CoV-2 HKG/HKU-904a/2020 strain (GenBank accession no. MT365032) with a query cover of 96% and sequence identity of 81.47%. The maximum-likelihood analysis with sarbecoviruses demonstrated that the full genome and spike protein (S) gene of Rc-o319 were positioned within a specific clade that included SARS-CoV-2 (Figure 1, panels A, B). Amino acid sequences of open reading frame 1ab (ORF1ab) and S of Rc-o319 also were positioned within the SARS-CoV-2 clade (Figure 1 panels C and D). The phylogenetic trees maintained the same topology between ORF1ab and S, indicating that no recombination event occurred in Rc-0319, which was supported by similarity plot analysis (Appendix Fig

Influenza D virus has been found to cause respiratory diseases in livestock. We surveyed healthy dromedary camels in Ethiopia and found a high seroprevalence for this virus, in contrast to animals co-existing with the camels. Our observation implies that dromedary camels may play an important role in the circulation of influenza D virus.

H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.

Influenza D virus (IDV) can potentially cause respiratory diseases in livestock. We isolated a new IDV strain from diseased cattle in Japan; this strain is phylogenetically and antigenically distinguished from the previously described IDVs.

Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3′-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures. IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.