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

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) 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.

Influenza (flu) D virus, a possible causative agent of bovine respiratory disease, is genetically classified into three clusters: D/OK-, D/660-, and D/Japan-lineages. To evaluate antigenic heterogeneity among these clusters, we compared antibody titers to each lineage virus using bovine sera collected over time following virus infection. Antibody titers to D/Japan-lineage virus rose rapidly in the acute phase of infection, and were 4 times higher than those to the other clustered viruses. In the later phase of infection, titers to D/Japan-lineage virus were equivalent to those to D/OK-lineage virus, and still higher than those to D/660-lineage virus. These results suggest the existence of common and lineage-specific antigenic epitopes in the hemagglutinin-esterase-fusion protein of flu D viruses.

250 words 15 Text 4,170 words 16 2 ABSTRACT Influenza D virus (IDV) was initially isolated in the USA in 2011. IDV is 17distributed worldwide and is one of the causative agents of bovine respiratory disease 18 complex (BRDC), which exhibits high morbidity and mortality in feedlot cattle. 19 Molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics 20 systems are vital tools not only for studying the biology of viruses, but also for use in 21 applications such as recombinant vaccine viruses. Here, we report the establishment of a 22 plasmid-based reverse genetics system for IDV. We first verified that the 3′-terminal 23 nucleotide of each 7-segmented genomic RNA contained uracil in contrary to the 24 previous report, and were then able to successfully generate recombinant IDV by 25 co-transfecting 7 plasmids containing these genomic RNAs along with 4 plasmids 26 expressing polymerase proteins and NP into HRT-18G cells. The recombinant virus had 27 a growth deficit compared to the wild-type virus, and we determined the reason for this 28 growth difference by examining the genomic RNA content of the viral particles. We 29 found that recombinant virus incorporated an unbalanced ratio of viral RNA segments 30 into particles as compared to the wild-type virus, and thus we adjusted the amount of 31 each plasmid used in transfection to obtain recombinant virus with the same replicative 32 capacity as wild-type virus. Our work here in establishing a reverse genetics system for 33 IDV will have a broad range of applications, including uses in studies focused on better 34 understanding IDV replication and pathogenicity as well as those contributing to the 35 development of BRDC countermeasures. 36 37 IMPORTANCE Bovine respiratory disease complex (BRDC) exhibits high mortality 38 and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is 39 considered to be a causative agent of BRDC. Here, we developed a reverse genetics 40 3 system that allows for the generation of IDV from cloned cDNAs, and the introduction 41 of mutations into the IDV genome. This reverse genetics system will become a 42 powerful tool for use in studies related to understanding the molecular mechanisms of 43 viral replication and pathogenicity, and will also lead to the development of new 44 countermeasures against BRDC. 45 46 KEYWORD bovine respiratory disease complex, influenza D virus, mutant, 47 recombinant virus, reverse genetics, transfection 48 49 50 Influenza D virus (IDV), a member of the family Orthomyxoviridae, was first isolated 52 from pigs with respiratory illness in Oklahoma, USA, in 2011 (1, 2). Epidemiological 53 analyses revealed that cattle are the main host of the virus, due to their high 54 seroprevalence for IDV (2, 3). Further epidemiological studies revealed that IDVs 55 circulate in cattle in many countries including the USA (2-4), Mexico (5), China (6), 56 Japan (7, 8), France (9), Italy (10), Ireland (11), Luxembourg (12), and African 57 countries (13). Furthermore, serolo...