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.

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.

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.

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.

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.

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.

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.

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.

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 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 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.

Pre-exposure with influenza A virus A/WSN/1933(H1N1) resulted in viral shedding reduction from pigs challenged with either swine H1N1 or H3N2 virus.

There is an urgent need to develop a broad-spectrum vaccine that can effectively prevent or eliminate the spread of co-circulating swine influenza virus strains in multiple lineages or subtypes. We describe here that pre-exposure with a live virus generated via a A/WSN/1933(H1N1) reverse genetics system resulted in a significant reduction of viral shedding from pigs exposed to either a swine H1N1 virus or a swine H3N2 virus. At 3-day post challenge (DPC), approximately 1 log and 1.5 logs reductions of viral shedding were observed in the swine H1N1- and H3N2-challenged vaccinated pigs when compared to unvaccinated animals. A further decline in viral load was observed at 5 DPC where viral shedding was decreased by greater than 3 logs in vaccinated pigs receiving either the H1N1 or H3N2 virus challenge. Although the sera of the vaccinated pigs contained high titers of neutralizing antibodies against the vaccine strain, measured by Hemagglutination Inhibition (HI) assay, only suboptimal HI titers of neutralizing antibody were detected in the post-challenge serum of the vaccinated animals using the challenge swine H1N1 virus. The substantial genetic and antigenic differences between the vaccine virus and the challenge viruses imply that the observed protection may be mediated by mechanisms other than neutralization by IgG, such as non-neutralizing antibody activities, mucosal immunity, or conserved T cell immunity, which warrants further investigation.

Detailed mapping of the linear B Cell epitopes of the hemagglutinin (HA) protein of swine influenza virus.

Using the Hemagglutinin (HA) protein peptide array of H1N1 pdm09 and a panel of swine antisera against various swine influenza H1 and H3 clusters, we identified three immunoreactive epitopes with one (peptide 15) located in HA1 (amino acids 57-71) and two (peptides 121 and 139) in HA2 (amino acids 481-495 and 553-566). Further analysis showed that all swine antisera of H1 clusters efficiently recognized two HA2 epitopes; peptides 121 and 139, with only a subset of antisera reactive to HA1-derived peptide 15. Interestingly, none of these peptides were reactive to SIV H3 antisera. Finally, intranasal inoculation of peptides 15 and 121 into pigs revealed that peptide 121, not peptide 15, was able to generate antibody responses in some animals. The results of our experiments provide an important foundation for further analyzing the immune response against these peptides during natural viral infection and also provide peptide substrates for diagnostic assays.

Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection.

Influenza viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses.

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin-Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs.

Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.