Antigenic commonality and divergence of hemagglutinin-esterase-fusion protein among influenza D virus lineages revealed using epitope mapping.

Influenza D virus (IDV) is one of the causative agents of bovine respiratory disease complex, which is the most common and economically burdensome disease affecting the cattle industry, and the need for an IDV vaccine has been proposed to enhance disease control. IDVs are classified into five genetic lineages based on the coding sequences of the hemagglutinin-esterase-fusion (HEF) protein, an envelope glycoprotein, which is the main target of protective antibodies against IDV infection. Herein, we prepared a panel of monoclonal antibodies (mAbs) against the HEF protein of viruses of various lineages to investigate the antigenic characteristics of IDVs and found that the mAbs could be largely separated into three groups. The first, second, and third groups demonstrated lineage-specific reactivity, cross-reactivity to viruses of multiple but not all lineages, and cross-reactivity to viruses of all lineages, respectively. Analyzing the escape mutant viruses from virus-neutralizing mAbs revealed that the receptor-binding region of the HEF molecule harbors virus-neutralizing epitopes that are conserved across multiple lineage viruses. In contrast, the apex region of the molecule possessed epitopes unique to each lineage virus. Furthermore, reverse genetics-generated recombinant viruses with point mutations revealed that amino acids within positions 210-214 of the HEF protein determined the antigenic specificity of each lineage virus. Taken together, this study reveals considerable antigenic variation among IDV lineages, although they are presumed to form a single serotype in terms of HEF antigenicity. Characterization of the antigenic epitope structure of HEF may contribute to selecting and creating effective vaccine viruses against IDV.IMPORTANCEInfluenza D viruses (IDVs) are suggested to create cross-reactive single serotypes in hemagglutinin-esterase-fusion (HEF) antigenicity, as indicated by serological analyses among distinct HEF lineage viruses. This is supported by the high identities of HEF gene sequences among strains, unlike the hemagglutinin (HA) genes of the influenza A virus that exhibit HA subtypes. Herein, we analyzed HEF antigenicity using a monoclonal antibody panel prepared from several virus lineages and found the existence of lineage-conserved and lineage-specific epitopes in HEF molecules. These findings confirm the HEF commonality and divergence among IDVs and provide useful information for constructing a vaccine containing a recombinant IDV virus with an engineered HEF gene, thereby leading to broad immunogenicity.

Influenza A Virus Agnostic Receptor Tropism Revealed Using a Novel Biological System with Terminal Sialic Acid Knockout Cells.

Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. In this study, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. IMPORTANCE The acquisition of mutations that allow avian influenza A virus hemagglutinins to recognize human-type receptors is mandatory for the transmission of avian viruses to humans, which could lead to a pandemic. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study contributes to elucidation of the relationship between the physical binding of virus and receptor and viral infectivity. Furthermore, the system using sialic acid knockout cells could provide a useful tool to explore the sialic acid-independent entry mechanism. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism.

Direct evidence of fiber-protein-directed hemagglutination by canine adenoviruses.

Canine adenoviruses (CAdVs) are divided into two serotypes, CAdV1 and CAdV2, whose members mainly cause infectious hepatitis and laryngotracheitis, respectively, in canids. To gain insight into the molecular basis of viral hemagglutination, we constructed chimeric viruses whose fiber proteins or their knob domains, which play a role in viral attachment to cells, were swapped among CAdV1, CAdV2, and bat adenovirus via reverse genetics. The results revealed that, in each case, viral hemagglutination was specifically mediated by the fiber protein or knob domain, providing direct evidence for fiber-protein-directed receptor-binding characteristics of CAdVs.

Isolation and genetic characterization of a mammalian orthoreovirus from Vespertilio sinensis in Japan.

Throughout East Asia, Europe, and North America, mammalian orthoreovirus (MRV), for which bats have been proposed to be natural reservoirs, has been detected in a variety of domestic and wild mammals, as well as in humans. Here, we isolated a novel MRV strain (designated as Kj22-33) from a fecal sample from Vespertilio sinensis bats in Japan. Strain Kj22-33 has a 10-segmented genome with a total length of 23,580 base pairs. Phylogenetic analysis indicated that Kj22-33 is a serotype 2 strain, the segmented genome of which has undergone reassortment with that of other MRV strains.

Differential role of NSs genes in the neurovirulence of two genogroups of Akabane virus causing postnatal encephalomyelitis.

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

Isolation of Bat Sarbecoviruses, Japan.

Surveillance of bat betacoronaviruses is crucial for understanding their spillover potential. We isolated bat sarbecoviruses from Rhinolophus cornutus bats in multiple locations in Japan. These viruses grew efficiently in cells expressing R. cornutus angiotensin converting enzyme-2, but not in cells expressing human angiotensin converting enzyme-2, suggesting a narrow host range.

Complete genome sequence of a novel bat mastadenovirus C strain isolated from Rhinolophus cornutus in Japan.

Here, we report a novel bat adenovirus strain isolated from apparently healthy bats of the species Rhinolophus cornutus in Japan. The genome of the isolate was 36,506 bp in length and encoded at least 33 proteins. Phylogenetic analysis of the DNA polymerase amino acid sequence, which provides one demarcation criterion for adenoviral species, indicated that the isolate belongs to the species Bat mastadenovirus C in the genus Mastadenovirus. Most of the encoded proteins shared high sequence similarity with those of known bat adenovirus C strains detected in different species of Rhinolophus, whereas the fiber protein and some E3- and E4-related proteins shared moderate similarity, and only the large E3 protein, which contains several host immune-suppression-related motifs, showed considerably lower similarity.

Establishment of a Reverse Genetics System for Influenza D Virus.

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 D Virus of New Phylogenetic Lineage, Japan.

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.

Detection and Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2, Japan.

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.

Influenza D Virus Infection in Dromedary Camels, Ethiopia.

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.

Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses.

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.

Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein.

UNLABELLED: We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized. IMPORTANCE: AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Japanese Encephalitis Virus and Schizophyllum commune Co-Infection in a Harbor Seal in Japan.

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a wide host range, extending from pigs and ardeid birds to opportunistic dead-end hosts, such as humans and horses. However, JEV encephalitis infections in aquatic mammals are rare, with only two cases in seals reported to date. Here, we report a lethal case of JEV and Schizophyllum commune co-infection in an aquarium-housed harbor seal in Japan. We isolated JEV from the brain of the dead seal and characterized its phylogeny and pathogenicity in mice. The virus isolate from the seal was classified as genotype GIb, which aligns with recent Japanese human and mosquito isolates as well as other seal viruses detected in China and Korea, and does not exhibit a unique sequence trait distinct from that of human and mosquito strains. We demonstrated that the seal isolate is pathogenic to mice and causes neuronal symptoms. These data suggest that seals should be considered a susceptible dead-end host for circulating JEV in natural settings.

Generation of a recombinant temperature-sensitive influenza D virus.

Influenza D virus (IDV) is a causative agent of the bovine respiratory disease complex (BRDC), which is the most common and costly disease affecting the cattle industry. For developing a candidate vaccine virus against IDV, we sought to produce a temperature-sensitive strain, similar to the live attenuated, cold-adapted vaccine strain available against the influenza A virus (IAV). To this end, we produced a recombinant IDV (designated rD/OK-AL) strain by introducing mutations responsible for the adaptation of the IAV vaccine strain to cold conditions and conferring sensitivity to high temperatures into PB2 and PB1 proteins using reverse genetics. The rD/OK-AL strain grew efficiently at 33 °C but did not grow at 37 °C in the cell culture, indicating its high-temperature sensitivity. In mice, rD/OK-AL was attenuated following intranasal inoculation. It mediated the production of high levels of antibodies against IDV in the serum. When the rD/OK-AL-inoculated mice were challenged with the wild-type virus, the virus was not detected in respiratory organs after the challenge, indicating complete protection against IDV. These results imply that the rD/OK-AL might be a potential candidate for the development of live attenuated vaccines for IDV that can be used to control BRDC.

Adaptation of the H7N2 Feline Influenza Virus to Human Respiratory Cell Culture.

During 2016-2017, the H7N2 feline influenza virus infected more than 500 cats in animal shelters in New York, USA. A veterinarian who had treated the cats became infected with this feline virus and showed mild respiratory symptoms. This suggests that the H7N2 feline influenza virus may evolve into a novel pandemic virus with a high pathogenicity and transmissibility as a result of mutations in humans. In this study, to gain insight into the molecular basis of the transmission of the feline virus to humans, we selected mutant viruses with enhanced growth in human respiratory A549 cells via successive passages of the virus and found almost all mutations to be in the envelope glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). The reverse genetics approach revealed that the HA mutations, HA1-H16Q, HA2-I47T, or HA2-Y119H, in the stalk region can lead to a high growth of mutant viruses in A549 cells, possibly by changing the pH threshold for membrane fusion. Furthermore, NA mutation, I28S/L, or three-amino-acid deletion in the transmembrane region can enhance viral growth in A549 cells, possibly by changing the HA-NA functional balance. These findings suggest that the H7N2 feline influenza virus has the potential to become a human pathogen by adapting to human respiratory cells, owing to the synergistic biological effect of the mutations in its envelope glycoproteins.

Different organ and tissue tropism between Akabane virus genogroups in a mouse model.

Akabane virus (AKAV) is an etiological agent that is teratogenic to the fetus of domestic ruminants, causing a significant loss of reproduction in livestock. In East Asia, AKAV isolates form two major clusters: genogroups I and II. In recent years, genogroup I isolates have also been associated with postnatal encephalomyelitis, mainly in calves. Here, we compared the pathogenicity in mice using genogroup I Iriki and genogroup II OBE-1 strains. Only mice infected intraperitoneally with the Iriki strain died and showed marked replication in the central nervous system (CNS) and lymphoid tissues. A more elevated blood-brain barrier (BBB) permeability was found in the Iriki-infected mice in the clinical phase, indicating that the BBB might be a possible route of viral transmission from the periphery to the CNS. These findings demonstrate that the Iriki strain presents greater neurovirulence and neuroinvasiveness compared with the OBE-1 strain, determining different AKAV pathogenicity among genogroups.

Detection and genetic characterization of bat MERS-related coronaviruses in Japan.

Betacoronaviruses, containing sarbecoviruses such as severe acute respiratory syndrome coronaviruses (SARS-CoV) and merbecovirus such as Middle East respiratory syndrome coronavirus (MERS-CoV), caused three human outbreaks in the past 2 decades; in particular, SARS-CoV-2 has caused the coronavirus disease 2019 pandemic. Since the ancestor of betacoronaviruses originated from wild bats, unidentified bat betacoronaviruses are presumed to be transmitted to humans in the future. In this study, we detected novel bat merbecoviruses from Vespertilio sinensis and Eptesicus japonensis, belonging to the family Vespertilionidae, in Japan. We found that these merbecoviruses were phylogenetically most closely related to the those previously detected in China. Alignment of the predicted receptor-binding motif on the spike proteins indicated that the Japanese bat merbecoviruses did not possess the specific amino acid residues that could be responsible for binding of MERS-CoV to the human dipeptidyl peptidase-4 receptor, which is unlikely to infect humans. This study demonstrated that bat merbecoviruses are widely conserved in multiple bat species of Vespertilionidae in East Asia, emphasizing the need for extensive epidemiological and biological studies on bat betacoronaviruses to facilitate the risk assessment of their spillover potential to humans.

Construction of an Influenza D Virus with an Eight-Segmented Genome.

Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the NS1 or NS2 genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.