Epidemiology of bat Betacoronavirus, subgenus Sarbecovirus is largely unknown, especially outside China. We detected a sarbecovirus phylogenetically related to severe acute respiratory syndrome coronavirus 2 from Rhinolophus cornutus bats in Japan. The sarbecovirus’ spike protein specifically recognizes angiotensin-converting enzyme 2 of R. cornutus, but not humans, as an entry receptor.

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.

Akabane virus (AKAV) and Schmallenberg virus (SBV) are members of the genus Orthobunyavirus , which are transmitted by arthropod vectors with a broad cellular tropism in vitro as well as in vivo . Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system and measured the quantities of binding of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least in vitro , to promote virus replication in susceptible cells. IMPORTANCE AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines.

Isolation of Bat Sarbecoviruses, Japan

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.