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.

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.

Japanese Macaques (Macaca fuscata) as Natural Reservoir of Bartonella quintana.

Bartonella quintana bacteremia was detected in 6 (13.3%) of 45 wild-caught Japanese macaques (Macaca fuscata). Multilocus sequence typing of the isolates revealed that Japanese macaques were infected with a new and specific B. quintana sequence type. Free-ranging Japanese macaques thus represent another natural reservoir of B. quintana.

Adaptation potential of H3N8 canine influenza virus in human respiratory cells.

In 2004, the equine-origin H3N8 canine influenza virus (CIV) first caused an outbreak with lethal cases in racing greyhounds in Florida, USA, and then spread to domestic dogs nationwide. Although transmission of this canine virus to humans has not been reported, it is important to evaluate its zoonotic potential because of the high contact opportunities between companion dogs and humans. To gain insight into the interspecies transmissibility of H3N8 CIV, we tested its adaptability to human respiratory A549 cells through successive passages. We found that CIV acquired high growth properties in these cells mainly through mutations in surface glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). Our reverse genetics approach revealed that HA2-K82E, HA2-R163K, and NA-S18L mutations were responsible for the increased growth of CIV in human cells. Molecular analyses revealed that both HA2 mutations altered the optimum pH for HA membrane fusion activity and that the NA mutation changed the HA-NA functional balance. These findings suggest that H3N8 CIV could evolve into a human pathogen with pandemic potential through a small number of mutations, thereby posing a threat to public health in the future.

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.

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.

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.

A potential bat adenovirus-based oncolytic virus targeting canine cancers.

Although a canine adenovirus (CAdV)-based oncolytic virus (OV) candidate targeting canine tumors has been reported, its oncolytic effect could be attenuated by CAdV vaccine-induced neutralizing antibodies in dog patients. To circumvent this issue, we focused on the bat adenovirus (BtAdV) strain, which was previously isolated from healthy microbats. We previously showed that this virus replicated efficiently in canine cell lines and did not serologically cross-react with CAdVs, suggesting that it may offer the possibility of an OV candidate for canine tumors. Here, we tested the growth properties and cytotoxicity of the BtAdV Mm32 strain in a panel of canine tumor cells and found that its characteristics were equivalent to those of CAdVs. To produce an Mm32 construct with enhanced tumor specificity, we established a novel reverse genetics system for BtAdV based on bacterial artificial chromosomes, and generated a recombinant virus, Mm32-E1Ap + cTERTp, by inserting a tumor-specific canine telomerase reverse transcriptase promoter into its E1A regulatory region. The growth and cytotoxicity of this recombinant were superior to those of wild-type Mm32 in canine tumor cells, unlike in normal canine cells. These data suggest that Mm32-E1Ap + cTERTp could be a promising OV for alternative canine cancer therapies.

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.

Adaptation of H3N2 canine influenza virus to feline cell culture.

H3N2 canine influenza viruses are prevalent in Asian and North American countries. During circulation of the viruses in dogs, these viruses are occasionally transmitted to cats. If this canine virus causes an epidemic in cats too, sporadic infections may occur in humans because of the close contact between these companion animals and humans, possibly triggering an emergence of mutant viruses with a pandemic potential. In this study, we aimed to gain an insight into the mutations responsible for inter-species transmission of H3N2 virus from dogs to cats. We found that feline CRFK cell-adapted viruses acquired several mutations in multiple genome segments. Among them, HA1-K299R, HA2-T107I, NA-L35R, and M2-W41C mutations individually increased virus growth in CRFK cells. With a combination of these mutations, virus growth further increased not only in CRFK cells but also in other feline fcwf-4 cells. Both HA1-K299R and HA2-T107I mutations increased thermal resistance of the viruses. In addition, HA2-T107I increased the pH requirement for membrane fusion. These findings suggest that the mutations, especially the two HA mutations, identified in this study, might be responsible for adaptation of H3N2 canine influenza viruses in cats.