RETRACTED: Skelton et al. Contribution of Host Immune Responses against Influenza D Virus Infection toward Secondary Bacterial Infection in a Mouse Model. Viruses 2019, 11, 994.

The Viruses Editorial Office retracts the article, "Contribution of Host Immune Responses Against Influenza D Virus Infection Toward Secondary Bacterial Infection in a Mouse Model" [...].

A recombinant chimeric influenza virus vaccine expressing the consensus H3 hemagglutinin elicits broad hemagglutination inhibition antibodies against divergent swine H3N2 influenza viruses.

The global distribution and ongoing evolution of type A swine influenza virus (IAV-S) continue to pose significant challenges against developing broadly protective vaccines to control swine influenza. This study focuses on the hemagglutinin (HA) consensus-based approach towards developing a more broadly protective swine influenza vaccine against various H3 strains circulating in domestic pig populations. By computationally analyzing >1000 swine H3 full-length HA sequences, we generated a consensus H3 and expressed it in the context of influenza A WSN/33 reverse genetics system. The derived recombinant chimeric swine influenza virus with the consensus H3 was inactivated and further evaluated as a potential universal vaccine in pigs. The consensus H3 vaccine elicited broadly active hemagglutination inhibition (HI) antibodies against divergent swine H3N2 influenza viruses including human H3N2 variant of concern, and strains belong to genetic clusters IV, IV-A, IV-B, IV-C, IV-D and IV-F. Importantly, vaccinated pigs were completely protected against challenge with a clinical swine H3N2 isolate in that neither viral shedding nor replication in lungs of vaccinated pigs were observed. These findings warrant further study of the consensus H3 vaccine platform for broad protection against diverse swine influenza viruses.

Prevalence and characterization of seven-segmented influenza viruses in bovine respiratory disease complex.

Bovine respiratory disease (BRD) complex is a multifactorial respiratory disease of cattle. Seven-segmented influenza C (ICV) and D (IDV) viruses have been identified in cattle with BRD, however, molecular epidemiology and prevalence of IDV and ICV in the diseased population remain poorly characterized. Here, we conducted a molecular screening of 208 lung samples of bovine pneumonia cases for the presence of IDV and ICV. Our results demonstrated that both viruses were prevalent in BRD cases and the overall positivity rates of IDV and ICV were 20.88% and 5.99% respectively. Further analysis of three IDV strains isolated from lungs of cattle with BRD showed that these lung-tropic strains belonged to D/Michigan/2019 clade and diverged antigenically from the circulating dominant IDV clades D/OK and D/660. Our results reveal that IDV and ICV are associated with BRD complex and support a role for IDV and ICV in the etiology of BRD.

A fully human monoclonal antibody possesses antibody-dependent cellular cytotoxicity (ADCC) activity against the H1 subtype of influenza A virus by targeting a conserved epitope at the HA1 protomer interface.

The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCC activity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.

Influenza C and D Viruses Demonstrated a Differential Respiratory Tissue Tropism in a Comparative Pathogenesis Study in Guinea Pigs.

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism.

Experimental Infection of Horses with Influenza D Virus.

Antibodies to influenza D virus (IDV) have been detected in horses, but no evidence of disease in the field has been reported. To determine whether IDV is infectious, immunogenic, and pathogenic in horses, four 2-year-old horses seronegative for both influenza A (H3N8) and D viruses were intranasally inoculated with 6.25 × 107 TCID50/animal of D/bovine/California/0363/2019 (D/CA2019) virus, using a portable equine nebulizer system. Horses were observed daily for clinical signs including rectal temperature, nasal discharge, coughing, lung sounds, tachycardia, and tachypnea. No horses exhibited clinical signs of disease. Nasopharyngeal swabs collected from 1-8 days post-infection demonstrated virus shedding by qRT-PCR. The horses showed evidence of seroconversion as early as 13 days post-infection (dpi) and the geometric mean of the antibody titers (GMT) of all four horses ranged from 16.82-160 as demonstrated by the microneutralization assay. Further, deep RNA sequencing of the virus isolated in embryonated chicken eggs revealed no adaptive mutations indicating that IDV can replicate in horses, suggesting the possibility of interspecies transmission of IDV with bovine reservoir into equids in nature.

Host Range, Biology, and Species Specificity of Seven-Segmented Influenza Viruses-A Comparative Review on Influenza C and D.

Other than genome structure, influenza C (ICV), and D (IDV) viruses with seven-segmented genomes are biologically different from the eight-segmented influenza A (IAV), and B (IBV) viruses concerning the presence of hemagglutinin-esterase fusion protein, which combines the function of hemagglutinin and neuraminidase responsible for receptor-binding, fusion, and receptor-destroying enzymatic activities, respectively. Whereas ICV with humans as primary hosts emerged nearly 74 years ago, IDV, a distant relative of ICV, was isolated in 2011, with bovines as the primary host. Despite its initial emergence in swine, IDV has turned out to be a transboundary bovine pathogen and a broader host range, similar to influenza A viruses (IAV). The receptor specificities of ICV and IDV determine the host range and the species specificity. The recent findings of the presence of the IDV genome in the human respiratory sample, and high traffic human environments indicate its public health significance. Conversely, the presence of ICV in pigs and cattle also raises the possibility of gene segment interactions/virus reassortment between ICV and IDV where these viruses co-exist. This review is a holistic approach to discuss the ecology of seven-segmented influenza viruses by focusing on what is known so far on the host range, seroepidemiology, biology, receptor, phylodynamics, species specificity, and cross-species transmission of the ICV and IDV.

Identification of a Ruminant Origin Group B Rotavirus Associated with Diarrhea Outbreaks in Foals.

Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.

Isolation and development of bovine primary respiratory cells as model to study influenza D virus infection.

Influenza D virus (IDV) is a novel type of influenza virus that infects and causes respiratory illness in bovines. Lack of host-specific in vitro model that can recapitulate morphology and physiology of in vivo airway epithelial cells has impeded the study of IDV infection. Here, we established and characterized bovine primary respiratory epithelial cells from nasal turbinate, soft palate, and trachea of the same calf. All three cell types showed characteristics peculiar of epithelial cells, polarized into apical-basolateral membrane, and formed tight junctions. Furthermore, these cells expressed both α-2,3- and α-2,6-linked sialic acids with α-2,3 linkage being more abundant. IDV strains replicated to high titers in these cells, while influenza A and B viruses exhibited moderate to low titers, with influenza C virus replication not detected. These findings suggest that bovine primary airway epithelial cells can be utilized to model infection biology and pathophysiology of IDV and other respiratory pathogens.

Emergence of new phylogenetic lineage of Influenza D virus with broad antigenicity in California, United States.

Influenza D virus (IDV), with bovines as a primary host, circulates widely in cattle populations across North America and Eurasia. Here we report the identification of a novel IDV group with broad antigenicity in U.S. bovine herds, which is genetically different from previously known lineages of IDV.

Piglet immunization with a spike subunit vaccine enhances disease by porcine epidemic diarrhea virus.

Immunization with an insect cell lysate/baculovirus mixture containing recombinant porcine epidemic diarrhea virus (PEDV) spike protein induced high levels of neutralizing antibodies in both mice and piglets. However, immunization of piglets with this vaccine resulted in enhancement of disease symptoms and virus replication in vaccine recipients exposed to PEDV challenge. Thus, these observations demonstrate a previously unrecognized challenge of PEDV vaccine research, which has important implications for coronavirus vaccine development.

Susceptibility of livestock and companion animals to COVID-19.

While the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to wreak havoc, there is little known about the susceptibility of the livestock and companion animals relative to humans. Here, we explore the susceptibility of companion and agricultural animals, in light of the existing information on natural infections, experimental infections, serosurveillance, and in vitro protein-homology binding interaction studies of the SARS-CoV-2 with the proposed receptor angiotensin-converting enzyme 2 from diverse animal species.

Human Monoclonal Antibody Derived from Transchromosomic Cattle Neutralizes Multiple H1 Clades of Influenza A Virus by Recognizing a Novel Conformational Epitope in the Hemagglutinin Head Domain.

Influenza remains a global health risk and challenge. Currently, neuraminidase (NA) inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug-resistant variants. Neutralizing antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. However, the major hurdle for the extensive application of antibody therapies lies in the difficulty of generating nonimmunogenic antibodies in large quantities rapidly. Here, we report that one human monoclonal antibody (MAb), 53C10, isolated from transchromosomic (Tc) cattle exhibits potent neutralization and hemagglutination inhibition titers against different clades of H1N1 subtype influenza A viruses. In vitro selection of antibody escape mutants revealed that 53C10 recognizes a novel noncontinuous epitope in the hemagglutinin (HA) head domain involving three amino acid residues, glycine (G), serine (S), and glutamic acid (E) at positions 172, 207, and 212, respectively. The results of our experiments supported a critical role for substitution of arginine at position 207 (S207R) in mediating resistance to 53C10, while substitutions at either G172E or E212A did not alter antibody recognition and neutralization. The E212A mutation may provide structural stability for the epitope, while the substitution G172E probably compensates for loss of fitness introduced by S207R. Our results offer novel insights into the mechanism of action of MAb 53C10 and indicate its potential role in therapeutic treatment of H1 influenza virus infection in humans.IMPORTANCE Respiratory diseases caused by influenza viruses still pose a serious concern to global health, and neutralizing antibodies constitute a promising area of antiviral therapeutics. However, the potential application of antibodies is often hampered by the challenge in generating nonimmunogenic antibodies in large scale. In the present study, transchromosomic (Tc) cattle were used for the generation of nonimmunogenic monoclonal antibodies (MAbs), and characterization of such MAbs revealed one monoclonal antibody, 53C10, exhibiting a potent neutralization activity against H1N1 influenza viruses. Further characterization of the neutralization escape mutant generated using this MAb showed that three amino acid substitutions in the HA head domain contributed to the resistance. These findings emphasize the importance of Tc cattle in the production of nonimmunogenic MAbs and highlight the potential of MAb 53C10 in the therapeutic application against H1 influenza virus infection in humans.

Influenza A Virus Antibodies with Antibody-Dependent Cellular Cytotoxicity Function.

Influenza causes millions of cases of hospitalizations annually and remains a public health concern on a global scale. Vaccines are developed and have proven to be the most effective countermeasures against influenza infection. Their efficacy has been largely evaluated by hemagglutinin inhibition (HI) titers exhibited by vaccine-induced neutralizing antibodies, which correlate fairly well with vaccine-conferred protection. Contrarily, non-neutralizing antibodies and their therapeutic potential are less well defined, yet, recent advances in anti-influenza antibody research indicate that non-neutralizing Fc-effector activities, especially antibody-dependent cellular cytotoxicity (ADCC), also serve as a critical mechanism in antibody-mediated anti-influenza host response. Monoclonal antibodies (mAbs) with Fc-effector activities have the potential for prophylactic and therapeutic treatment of influenza infection. Inducing mAbs mediated Fc-effector functions could be a complementary or alternative approach to the existing neutralizing antibody-based prevention and therapy. This review mainly discusses recent advances in Fc-effector functions, especially ADCC and their potential role in influenza countermeasures. Considering the complexity of anti-influenza approaches, future vaccines may need a cocktail of immunogens in order to elicit antibodies with broad-spectrum protection via multiple protective mechanisms.

Influenza D virus diverges from its related influenza C virus in the recognition of 9-O-acetylated N-acetyl- or N-glycolyl-neuraminic acid-containing glycan receptors.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other mammalian hosts. By using traditional hemagglutination assay coupled with sialoglycan microarray (SGM) platform and functional assays, we demonstrated that IDV is more efficient in recognizing both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) than influenza C virus (ICV), a ubiquitous human pathogen. ICV seems to strongly prefer Neu5,9Ac2 over Neu5Gc9Ac. Since Neu5Gc9Ac is different from Neu5,9Ac2 only by an additional oxygen in the group at the C5 position, our results reveal that the hydroxyl group in Neu5Gc9Ac plays a critical role in determining receptor binding specificity, which as a result may discriminate IDV from ICV in communicating with 9-O-acetylated SAs. These findings shall provide a framework for further investigation towards better understanding of how newly discovered multiple-species-infecting IDV exploits natural 9-O-acetylated SA variations to expand its host range.

Genetic and antigenic characteristics of a human influenza C virus clinical isolate.

Unlike influenza A and B viruses that infect humans and cause severe diseases in seasonal epidemics, influenza C virus (ICV) is a ubiquitous childhood pathogen typically causing mild respiratory symptoms. ICV infections are rarely diagnosed and less research has been performed on it despite the virus being capable of causing severe disease in infants. Here we report on the isolation of a human ICV from a child with acute respiratory disease, provisionally designated C/Victoria/2/2012 (C/Vic). The full-length genome sequence and phylogenetic analysis revealed that the hemagglutinin-esterase-fusion (HEF) gene of C/Vic was derived from C/Sao Paulo lineage, while its PB2 and P3 genes evolved separately from all characterized historical ICV isolates. Furthermore, antigenic analysis using the hemagglutination inhibition (HI) assay found that 1947 C/Taylor virus (C/Taylor lineage) was antigenically more divergent from1966 C/Johannesburg (C/Aichi lineage) than from 2012 C/Vic. Structure modeling of the HEF protein identified two mutations in the 170-loop of the HEF protein around the receptor-binding pocket as a possible antigenic determinant responsible for the discrepant HI results. Taken together, results of our studies reveal novel insights into the genetic and antigenic evolution of ICV and provide a framework for further investigation of its molecular determinants of antigenic property and replication.

Contribution of Host Immune Responses Against Influenza D Virus Infection Toward Secondary Bacterial Infection in a Mouse Model.

Influenza D viruses (IDV) are known to co-circulate with viral and bacterial pathogens in cattle and other ruminants. Currently, there is limited knowledge regarding host responses to IDV infection and whether IDV infection affects host susceptibility to secondary bacterial infections. To begin to address this gap in knowledge, the current study utilized a combination of in vivo and in vitro approaches to evaluate host cellular responses against primary IDV infection and secondary bacterial infection with Staphylococcus aureus (S. aureus). Primary IDV infection in mice did not result in clinical signs of disease and it did not enhance the susceptibility to secondary S. aureus infection. Rather, IDV infection appeared to protect mice from the usual clinical features of secondary bacterial infection, as demonstrated by improved weight loss, survival, and recovery when compared to S. aureus infection alone. We found a notable increase in IFN-ß expression following IDV infection while utilizing human alveolar epithelial A549 cells to analyze early anti-viral responses to IDV infection. These results demonstrate for the first time that IDV infection does not increase the susceptibility to secondary bacterial infection with S. aureus, with evidence that anti-viral immune responses during IDV infection might protect the host against these potentially deadly outcomes.

Influenza A in Bovine Species: A Narrative Literature Review.

It is quite intriguing that bovines were largely unaffected by influenza A, even though most of the domesticated and wild animals/birds at the human-animal interface succumbed to infection over the past few decades. Influenza A occurs on a very infrequent basis in bovine species and hence bovines were not considered to be susceptible hosts for influenza until the emergence of influenza D. This review describes a multifaceted chronological review of literature on influenza in cattle which comprises mainly of the natural infections/outbreaks, experimental studies, and pathological and seroepidemiological aspects of influenza A that have occurred in the past. The review also sheds light on the bovine models used in vitro and in vivo for influenza-related studies over recent years. Despite a few natural cases in the mid-twentieth century and seroprevalence of human, swine, and avian influenza viruses in bovines, the evolution and host adaptation of influenza A virus (IAV) in this species suffered a serious hindrance until the novel influenza D virus (IDV) emerged recently in cattle across the world. Supposedly, certain bovine host factors, particularly some serum components and secretory proteins, were reported to have anti-influenza properties, which could be an attributing factor for the resilient nature of bovines to IAV. Further studies are needed to identify the host-specific factors contributing to the differential pathogenetic mechanisms and disease progression of IAV in bovines compared to other susceptible mammalian hosts.

Development and characterization of a stable bovine intestinal sub-epithelial myofibroblast cell line from ileum of a young calf.

Intestinal sub-epithelial myofibroblasts (ISEMFs) are mesenchymal cells that do not express cytokeratin but express α-smooth muscle actin and vimentin. Despite being cells with diverse functions, there is a paucity of knowledge about their origin and functions primarily due to the absence of a stable cell line. Although myofibroblast in vitro models for human, mouse, and pig are available, there is no ISEMF cell line available from young calves. We isolated and developed an ileal ISEMF cell line from a 2-d-old calf that expressed α-smooth muscle actin and vimentin but no cytokeratin indicating true myofibroblast cells. To overcome replicative senescence, we immortalized primary cells with SV40 large T antigen. We characterized and compared both primary and immortalized ileal ISEMF cells for surface glycan and Toll-like-receptor (TLR) expression by lectin-binding assay and real-time quantitative PCR (RT-qPCR) assay respectively. SV40 immortalization significantly decreased surface lectin binding for lectins GSL-I, PHA-L, ECL, Jacalin, Con-A, LCA, and LEL. Both cell types expressed TLRs 1-9 and showed no significant differences in TLR expression. Thus, these cells can be useful in vitro model to study ISEMF's origin, physiology, and functions.

Development and characterization of swine primary respiratory epithelial cells and their susceptibility to infection by four influenza virus types.

Influenza viruses are a group of respiratory pathogens that have evolved into four different types: A, B, C, and D. A common feature is that all four types are capable of replicating and transmitting among pigs. Here, we describe the development of isogenous cell culture system from the swine respiratory tract to study influenza viruses. Phenotypic characterization of swine primary nasal turbinate, trachea and lung cells revealed high expression of cytokeratin and demonstrated tissue site dependent expression of tight junction proteins. Furthermore, lectin binding assay on these cells demonstrated higher levels of Sia2-6Gal than Sia2-3Gal receptors and supported the replication of influenza A, B, C, and D viruses to appreciable levels at both 33 and 37 °C, but replication competence was dependent on virus type or temperature used. Overall, these swine primary respiratory cells showed epithelial phenotype, which is suitable for studying the comparative biology and pathobiology of influenza viruses.