Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination.

Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed ß-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.

Receptor binding and immunogenic properties of the receptor binding domain of influenza D virus hemagglutinin-esterase-fusion protein expressed from Escherichia coli.

The hemagglutinin-esterase-fusion (HEF) protein binds 9-O-acetylated sialic acids-containing glycans on the cell surface and drives influenza D virus (IDV) entry. The HEF is a primary determinant of the exceptional thermal and acid stability observed in IDV infection biology. Here, we expressed and purified the receptor binding domain (RBD) of the IDV HEF protein in Escherichia coli and characterized its receptor binding and antigenic properties. The data from these experiments indicate that (i) the RBD can bind with specificity to turkey red blood cells (RBC), and its binding can be specifically inhibited by IDV antibody; (ii) the RBD efficiently binds to the cell surface of MDCK cells expressing the receptor of IDV; and (iii) anti-RBD antibodies are capable of blocking RBD attachment to MDCK cells as well as of inhibiting the virus from agglutinating RBCs. These observations support the utility of this RBD in future receptor and entry studies of IDV.

Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles.

Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization test is the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particles incorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface. Overall, this simple, safe, and rapid method could be used to detect MV NAbs.

Vaccine-induced neutralizing antibodies bind to the H protein of a historical measles virus.

Measles is a highly contagious airborne viral disease. It can lead to serious complications and death and is preventable by vaccination. The live-attenuated measles vaccine (LAMV) derived from a measles virus (MV) isolated in 1954 has been in use globally for six decades and protects effectively by providing a durable humoral and cell-mediated immunity. Our study addresses the temporal stability of epitopes on the viral surface glycoprotein hemagglutinin (H) which is the major target of MV-neutralizing antibodies. We investigated the binding of seven vaccine-induced MV-H-specific monoclonal antibodies (mAbs) to cell-free synthesized MV-H proteins derived from the H gene sequences obtained from a lung specimen of a fatal case of measles pneumonia in 1912 and an isolate from a current case. The binding of four out of seven mAbs to the H protein of both MV strains provides evidence of epitopes that are stable for more than 100 years. The binding of the universally neutralizing mAbs RKI-MV-12b and RKI-MV-34c to the H protein of the 1912 MV suggests the long-term stability of highly conserved epitopes on the MV surface.

Molecular Mechanisms behind Conformational Transitions of the Influenza Virus Hemagglutinin Membrane Anchor.

Membrane fusion is a fundamental process that is exploited by enveloped viruses to enter host cells. In the case of the influenza virus, fusion is facilitated by the trimeric viral hemagglutinin protein (HA). So far, major focus has been put on its N-terminal fusion peptides, which are directly responsible for fusion initiation. A growing body of evidence points also to a significant functional role of the HA C-terminal domain, which however remains incompletely understood. Our computational study aimed to elucidate the structural and functional interdependencies within the HA C-terminal region encompassing the transmembrane domain (TMD) and the cytoplasmic tail (CT). In particular, we were interested in the conformational shift of the TMD in response to varying cholesterol concentration in the viral membrane and in its modulation by the presence of CT. Using free-energy calculations based on atomistic molecular dynamics simulations, we characterized transitions between straight and tilted metastable TMD configurations under varying conditions. We found that the presence of CT is essential for achieving a stable, highly tilted TMD configuration. As we demonstrate, such a configuration of HA membrane anchor likely supports the tilting motion of its ectodomain, which needs to be executed during membrane fusion. This finding highlights the functional role of, so far, the relatively overlooked CT region.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Ferret model to mimic the sequential exposure of humans to historical H3N2 influenza viruses.

Mutations accumulate in influenza A virus proteins, especially in the main epitopes on the virus surface glycoprotein hemagglutinin (HA). For influenza A(H3N2) viruses, in particular, the antigenicity of their HA has altered since their emergence in 1968, requiring changes of vaccine strains every few years. Most adults have been exposed to several antigenically divergent H3N2 viruses through infection and/or vaccination, and those exposures affect the immune responses of those individuals. However, animal models reflecting this 'immune history' in humans are lacking and naïve animals are generally used for vaccination and virus challenge studies. Here, we describe a ferret model to mimic the serial exposure of humans to antigenically different historical H3HA proteins. In this model, ferrets were sequentially immunized with adjuvanted recombinant H3HA proteins from two or three different H3HA antigenic clusters in chronological order, and serum neutralizing antibody titers were examined against the homologous virus and viruses from different antigenic clusters. For ferrets immunized with a single HA antigen, serum neutralizing antibody titers were elevated specifically against the homologous virus. However, after immunization with the second or third antigenically distinct HA antigen in chronological order, the ferrets showed an increase in more broadly cross-reactive neutralizing titers against the antigenically distinct viruses and against the homologous virus. Sequentially immunized animals challenged with an antigenically advanced H3N2 virus showed attenuated virus growth and less body temperature increase compared with naïve animals. These results suggest that sequential exposure to antigenically different HAs elicits broader neutralizing activity in sera and enhances immune responses against more antigenically distinct viruses Our findings may partly explain why adults who have been exposed to antigenically divergent HAs are less likely to be infected with influenza virus and have severe symptoms than children.

Oral or intranasal immunization with recombinant Lactobacillus plantarum displaying head domain of Swine Influenza A virus hemagglutinin protects mice from H1N1 virus.

BACKGROUND: Swine influenza A virus (swIAV) is a major concern for the swine industry owing to its highly contagious nature and acute viral disease. Currently, most commercial swIAV vaccines are traditional inactivated virus vaccines. The Lactobacillus plantarum-based vaccine platform is a promising approach for mucosal vaccine development. Oral and intranasal immunisations have the potential to induce a mucosal immune response, which confers protective immunity. The aim of this study was to evaluate the probiotic potential and adhesion ability of three L. plantarum strains. Furthermore, a recombinant L. plantarum strain expressing the head domain of swIAV antigen HA1 was constructed and evaluated for its ability to prevent swIAV infection. RESULTS: The three L. plantarum strains isolated from healthy pig faecal samples maintained the highest survival rate when incubated at pH 3 and at bile salt concentration of 0.3%. They also showed high adherence to intestinal cells. All three L. plantarum strains were monitored in live mice, and no major differences in transit time were observed. Recombinant L. plantarum expressed swIAV HA1 protein (pSIP401-HA1-ZN-3) and conferred effective mucosal, cellular and systemic immune responses in the intestine as well as in the upper respiratory airways of mice. In conclusion, the oral and intranasal administration of L. plantarum strain pSIP401-HA1-ZN-3 in mice induced mucosal immunity and most importantly, provided protection against lethal influenza virus challenge. CONCLUSION: In summary, these findings suggest that the engineered L. plantarum strain pSIP401-HA1-ZN-3 can be considered as an alternative approach for developing a novel vaccine during an swine influenza A pandemic.

Receptor-Binding Specificity of Influenza Viruses.

Influenza A virus infection begins with the attachment of virus particles to sialic acid-containing receptors on the surface of host cells. This attachment is mediated by the viral surface glycoprotein hemagglutinin (HA). Influenza A viruses have a wide host range, meaning they are able to infect many mammal and bird species. Influenza pandemics have been caused by viruses that contain genes from avian influenza viruses. Therefore, the infection of humans with avian influenza viruses, including avian H5Nx and H7Nx viruses, poses a huge threat to public health. These avian influenza viruses can transmit directly to humans from infected poultry, but do not spread easily among people, in part, due to differences in the receptor-binding specificities of human and avian influenza viruses. Therefore, conversion from avian- to human-type receptor-binding specificity is widely believed to be necessary for the efficient transmission of avian influenza viruses among humans. Accordingly, constant monitoring of the receptor-binding specificity of avian influenza viruses is important. In this chapter, we describe the protocol for assessing the receptor-binding specificity of influenza A viruses.

Roles of Glycans and Non-glycans on the Epithelium and in the Immune System in H1-H18 Influenza A Virus Infections.

The large variation of influenza A viruses (IAVs) in various susceptible hosts and their rapid evolution, which allows host/tissue switching, host immune escape, vaccine escape, and drug resistance, are difficult challenges for influenza control in all countries worldwide. Access and binding of the IAV to actual receptors at endocytic sites is critical for the establishment of influenza infection. In this chapter, the progress in identification of and roles of glycans and non-glycans on the epithelium and in the immune system in H1-H18 IAV infections are reviewed. The first part of the review is on current knowledge of H1-H16 IAV receptors on the epithelium including sialyl glycans, other negatively charged glycans, and annexins. The second part of the review focuses on H1-H16 IAV receptors in the immune system including acidic surfactant phospholipids, Sia on surfactant proteins, the carbohydrate recognition domain (CRD) of surfactant proteins, Sia on mucins, Sia and C-type lectins on macrophages and dendritic cells, and Sia on NK cells. The third part of the review is about a possible H17-H18 IAV receptor. Binding of these receptors to IAVs may result in inhibition or enhancement of IAV infection depending on their location, host cell type, and IAV strain. Among these receptors, host sialyl glycans are key determinants of viral hemagglutinin (HA) lectins for H1-H16 infections. HA must acquire mutations to bind to sialyl glycans that are dominant on a new target tissue when switching to a new host for efficient transmission and to bind to long sialyl glycans found in the case of seasonal HAs with multiple glycosylation sites as a consequence of immune evasion. Although sialyl receptors/C-type lectins on immune cells are decoy receptors/pathogen recognition receptors for capturing viral HA lectin/glycans protecting HA antigenic sites, some IAV strains do not escape, such as by release with neuraminidase, but hijack these molecules to gain entry and replication in immune cells. An understanding of the virus-host battle tactics at the receptor level might lead to the establishment of novel strategies for effective control of influenza.

Development and applications of sialoglycan-recognizing probes (SGRPs) with defined specificities: exploring the dynamic mammalian sialoglycome.

Glycans that are abundantly displayed on vertebrate cell surface and secreted molecules are often capped with terminal sialic acids (Sias). These diverse 9-carbon-backbone monosaccharides are involved in numerous intrinsic biological processes. They also interact with commensals and pathogens, while undergoing dynamic changes in time and space, often influenced by environmental conditions. However, most of this sialoglycan complexity and variation remains poorly characterized by conventional techniques, which often tend to destroy or overlook crucial aspects of Sia diversity and/or fail to elucidate native structures in biological systems, i.e. in the intact sialome. To date, in situ detection and analysis of sialoglycans has largely relied on the use of plant lectins, sialidases, or antibodies, whose preferences (with certain exceptions) are limited and/or uncertain. We took advantage of naturally evolved microbial molecules (bacterial adhesins, toxin subunits, and viral hemagglutinin-esterases) that recognize sialoglycans with defined specificity to delineate 9 classes of sialoglycan recognizing probes (SGRPs: SGRP1-SGRP9) that can be used to explore mammalian sialome changes in a simple and systematic manner, using techniques common in most laboratories. SGRP candidates with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding nonbinding mutant probe as a simple and reliable negative control. The optimized panel of SGRPs can be used in methods commonly available in most bioscience labs, such as ELISA, western blot, flow cytometry, and histochemistry. To demonstrate the utility of this approach, we provide examples of sialoglycome differences in tissues from C57BL/6 wild-type mice and human-like Cmah-/- mice.

Biotechnological production of sialylated solid lipid microparticles as inhibitors of influenza A virus infection.

Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in 3 steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus.

A Deadly Embrace: Hemagglutination Mediated by SARS-CoV-2 Spike Protein at Its 22 N-Glycosylation Sites, Red Blood Cell Surface Sialoglycoproteins, and Antibody.

Rouleaux (stacked clumps) of red blood cells (RBCs) observed in the blood of COVID-19 patients in three studies call attention to the properties of several enveloped virus strains dating back to seminal findings of the 1940s. For COVID-19, key such properties are: (1) SARS-CoV-2 binds to RBCs in vitro and also in the blood of COVID-19 patients; (2) although ACE2 is its target for viral fusion and replication, SARS-CoV-2 initially attaches to sialic acid (SA) terminal moieties on host cell membranes via glycans on its spike protein; (3) certain enveloped viruses express hemagglutinin esterase (HE), an enzyme that releases these glycan-mediated bindings to host cells, which is expressed among betacoronaviruses in the common cold strains but not the virulent strains, SARS-CoV, SARS-CoV-2 and MERS. The arrangement and chemical composition of the glycans at the 22 N-glycosylation sites of SARS-CoV-2 spike protein and those at the sialoglycoprotein coating of RBCs allow exploration of specifics as to how virally induced RBC clumping may form. The in vitro and clinical testing of these possibilities can be sharpened by the incorporation of an existing anti-COVID-19 therapeutic that has been found in silico to competitively bind to multiple glycans on SARS-CoV-2 spike protein.

Hemagglutinating properties of a Streptococcus gordonii strain expressing sialic acid-binding adhesin homolog with low binding site similarity to that of strain DL1.

OBJECTIVES: The Hsa adhesin of Streptococcus gordonii strain DL1 was previously identified as a hemagglutinin that binds specifically to sialoglycoconjugates. We recently found that among oral streptococcal species, S. gordonii strains most frequently express Hsa homologs on the bacterial cell surface. However, the effect of amino acid sequence diversity of nonrepetitive region 2 (NR2), a putative binding site of Hsa, on antigenicity and hemagglutinating (HA) properties is unclear due to difficulties in DNA sequencing the NR2 coding region. The aim of this study was to elucidate the similarity of the low NR2 antigenicity Hsa homolog of strain NDU1118 to that of strain DL1 and the association of the homolog with HA properties of the strain. METHODS: The hsa homolog of NDU1118 was sequenced using a long-read next-generation sequencer, and the Hsa homolog was assessed by alignment analysis of the deduced amino acid sequences. The hsa mutant of NDU1118 was generated by insertion of the erythromycin resistance gene. The HA properties of the wild type and the hsa mutant were assessed with human erythrocytes. RESULTS: The NR2 amino acid sequence of the NDU1118 Hsa homolog was almost identical to that of the S. gordonii M99 Hsa homolog, also known as GspB, and less similar to that of DL1 Hsa. The hsa mutation of NDU1118 induced reduction of HA activity in untreated erythrocytes, but surprisingly increased lactose-inhibitable HA activity in neuraminidase-treated erythrocytes. CONCLUSIONS: The results suggest the existence of an adhesin other than the Hsa homolog on the cell surface of NDU1118.

Characterization of Influenza D Virus in Danish Calves.

Influenza D virus (IDV) was first described in 2011 and has been found to mainly circulate among cattle and swine populations worldwide. Nasal swab samples were collected from 100 Danish calf herds (83 dairy and 17 veal herds) from 2018-2020. Influenza D virus was detected in 12 of the herds. Samples with the lowest cycle quantification value were selected for full genome sequencing. A hemagglutinin-esterase fusion (HEF) gene sequence from a Danish IDV collected in 2015 was also included in this study. Phylogenetic analysis showed that viruses from seven of the IDV-positive herds belonged to the D/OK lineage and clustered together in the HEF tree with the IDV collected in 2015. Viruses from the four other herds belonged to the D/660 lineage, where three of the viruses clustered closely together, while the fourth virus was more phylogenetically distant in all gene segments. The high level of genetic similarity between viruses from two different herds involved in calf trading suggests that transmission occurred through the movement of calves. This study is, to our knowledge, the first to describe the characterization of IDV in calves in Denmark.

Self-assembling ferritin nanoparticles coupled with linear sequences from canine distemper virus haemagglutinin protein elicit robust immune responses.

BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.

Molecular and Antigenic Characterization of Avian H9N2 Viruses in Southern China.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10-3 substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 × 10-3 substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

A new canine distemper virus lineage identified from red pandas in China.

Canine distemper virus (CDV) is a highly contagious virus that causes multi-systemic, sub-clinical to fatal diseases in a wide range of carnivore species. Based on the sequences of the haemagglutinin (H) gene, CDV strains have been classified into 18 major genetic lineages. In this study, we characterized the genomes of CDV isolated from the lungs of two dead red pandas in China. Histopathological and immunohistochemical analyses revealed damage due to viral infection in these lungs. The two strains showed a deep genetic distance from the other 18 recognized lineages (>4.6% at nucleotide level and >5.0% at amino acid level). The maximum clade credibility tree of the H- gene sequences showed that they belonged to an independent clade and had diverged a relatively long time ago from the Asia-4 lineage (since 1884). These results suggest that the analyzed strains belong to a new CDV lineage, which we designate as Asia-6. Our finding indicates that CDV infections in wildlife in China are complex and are a threat to endangered carnivores.

Temporal and Gene Reassortment Analysis of Influenza C Virus Outbreaks in Hong Kong, SAR, China.

From 2014 to week 07/2020 the Centre for Health Protection in Hong Kong conducted screening for influenza C virus (ICV). A retrospective analysis of ICV detections to week 26/2019 revealed persistent low-level circulation with outbreaks occurring biennially in the winters of 2015 to 2016 and 2017 to 2018 (R. S. Daniels et al., J Virol 94:e01051-20, 2020, https://doi.org/10.1128/JVI.01051-20). Here, we report on an outbreak occurring in 2019 to 2020, reinforcing the observation of biennial seasonality in Hong Kong. All three outbreaks occurred in similar time frames, were subsequently dwarfed by seasonal epidemics of influenza types A and B, and were caused by similar proportions of C/Kanagawa/1/76 (K)-lineage and C/São Paulo/378/82 S1- and S2-sublineage viruses. Ongoing genetic drift was observed in all genes, with some evidence of amino acid substitution in the hemagglutinin-esterase-fusion (HEF) glycoprotein possibly associated with antigenic drift. A total of 61 ICV genomes covering the three outbreaks were analyzed for reassortment, and 9 different reassortant constellations were identified, 1 K-lineage, 4 S1-sublineage, and 4 S2-sublineage, with 6 of these being identified first in the 2019-1920 outbreak (2 S2-lineage and 4 S1-lineage). The roles that virus interference/enhancement, ICV persistent infection, genome evolution, and reassortment might play in the observed seasonality of ICV in Hong Kong are discussed. IMPORTANCE Influenza C virus (ICV) infection of humans is common, with the great majority of people being infected during childhood, though reinfection can occur throughout life. While infection normally results in "cold-like" symptoms, severe disease cases have been reported in recent years. However, knowledge of ICV is limited due to poor systematic surveillance and an inability to propagate the virus in large amounts in the laboratory. Following recent systematic surveillance in Hong Kong SAR, China, and direct ICV gene sequencing from clinical specimens, a 2-year cycle of disease outbreaks (epidemics) has been identified, with gene mixing playing a significant role in ICV evolution. Studies like those reported here are key to developing an understanding of the impact of influenza C virus infection in humans, notably where comorbidities exist and severe respiratory disease can develop.