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Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV and IBV, respectively). It has a monobasic site, which is cleaved by some host enzymes. The cleavage is essential to activating the virus, but the enzyme or enzymes in the respiratory tract have not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK cells (MDCK-TMPRSS2 and MDCK-HAT). ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multistep growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs for ICV infection. IMPORTANCE Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, which possesses receptor-binding, receptor-destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme or enzymes in the respiratory tract have not been identified. This study revealed that transmembrane protease serine S1 member 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of ICV infection.

Bovine coronavirus (BCoV) is the causative agent of diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract illnesses in cattle across the world. In this study, a total of 190 faecal samples from dairy calves with diarrhoea were collected from 14 farms in six Chinese provinces, and BCoV was detected in 18.95% (36/190) of the samples by reverse transcriptase polymerase chain reaction. Full‐length spike, hemagglutinin/esterase (HE), nucleocapsid and transmembrane genes were simultaneously cloned from 13 clinical samples (eight farms in four provinces), and most of the BCoV strains showed a unique evolutionary pattern based on the phylogenetic analysis of these genes. Interesting, 10 of the 13 strains were identified as HE recombinant strains, and these strains had experienced the same recombination event and carried the same recombination sites located between the esterase and lectin domain. They also shared an identical aa variant (F181V) in the R2‐loop. Moreover, 9/10 strains displayed another identical aa variant (P, S158A) in the adjacent R1‐loop of the HE gene, which differs from the other available BCoV HE sequences in the GenBank database. Our results showed that BCoV is widely circulating in dairy cattle in China, contributing to the diagnosis and control of dairy calves diarrhoea. Furthermore, a BCoV strain that carries a recombinant HE gene has spread in dairy calves in China. To the best of our knowledge, this is the first description of an HE recombination event occurring in BCoV; this is also the first description of the molecular prevalence of BCoV in China. Our findings will enhance current understanding about the genetic evolution of BCoV.

The codon usage pattern can reveal the adaptive changes that allow virus survival and fitness adaptation to their particular host, as well as the external environment. Although still considered a novel influenza virus, there is an increasing number of influenza D viruses (IDVs) reported. Considering the vital role of the hemagglutinin-esterase fusion (HEF) gene in receptor binding, receptor degradation, and membrane fusion, we investigated the codon usage pattern of the IDV HEF gene to better understand its adaptive changes during evolution. Based on the HEF gene, three groups including, D/OK, D/660, and D/Japan were identified. We found a low codon usage bias, which allowed IDV to replicate in the corresponding hosts by reducing competition during evolution, that was mainly driven by natural selection and mutation pressure, with a profound role of natural selection. Furthermore, the interaction between the codon adaption index (CAI) and the relative codon deoptimization index (RCDI) revealed the adaption of IDV to multiple hosts, especially cattle which is currently considered its reservoir. Additionally, similarity index (SiD) analysis revealed that the swine exerted a stronger evolutionary pressure on IDV than cattle, though cattle is considered the primary reservoir. In addition, the conserved PB1 gene showed a similar pattern of codon usage compared to HEF. Therefore, we hypothesized that IDV has a preference to maintain infection in multiple hosts. The study aids the understanding of the evolutionary changes of IDV, which could assist this novel virus prevention and control.

Graphic abstract We read with interest the article by Patel et al. on the identification of potential inhibitors of coronavirus hemagglutinin-esterase. The authors considered hemagglutinin-esterase as a glycoprotein of SARS-CoV-2 and selected hemagglutinin-esterase as a target to identify potential inhibitors using a combination of various computational approaches, and however, SARS-CoV-2 genome lacks hemagglutinin-esterase gene; thus, hemagglutinin-esterase does not exist in SARS-CoV-2 particle.

BACKGROUND: COVID-19 is not fully known and causes severe inflammation and cytokine storm. It has many symptoms, such as: fever, sore throat, headache, dyspnoea, and diarrhoea. Arbidol was used in the treatment of COVID19, which was the most critical health problem in the world. However, the desired recovery was not achieved with Arbidol. Many countries still use this drug in the treatment of COVID19. AIM: We aimed to determine whether Arbidol, the hemagglutinin esterase inhibitor used in the treatment of COVID-19, was effective against SARS Cov-2 in silico. RESULTS AND CONCLUSION: The similarity between hemagglutinin and spike proteins were reported due to the fact that inhibition properties of Arbidol and its 39 analogues were examined in detail against hemagglutinin esterase and spike glycoproteins. CID 1070884 and CID 1207786 were found to be more active against hemagglutinin esterase than in Arbidol, while these compounds were inactive against spike glycoproteins. The interaction mechanism was clarified between arbidol and spike proteins. Phenylalanine, tyrosine, glycine, lysine, and aspartic acid were found to be the headliner amino acids in the interactions between Arbidol and binding domains of spike glycoproteins in the SARS-CoV2 (Tab. 3, Fig. 8, Ref. 28).

The hemagglutinin-esterase (HE) protein of betacoronavirus lineage A is a secondary receptor in the infection process and is involved in the emergence of new betacoronavirus genotypes with altered host specificity and tissue tropism. We previously reported a novel recombinant bovine coronavirus (BCoV) strain that was circulating in dairy cattle in China, but this virus was not successfully isolated, and the genetic characteristics of BCoV are still largely unknown. In this study, 20 diarrheic faecal samples were collected from a farm in Liaoning province that had an outbreak of calf diarrhea (≤ 3 months of age) in November 2018, and all of the samples tested positive for BCoV by RT-PCR. In addition, a BCoV strain with a recombinant HE (designated as SWUN/A1/2018) and another BCoV strain with a recombinant HE containing an insertion (designated as SWUN/A10/2018) were successfully isolated in cell culture (TCID50: 104.25/mL and 104.73/mL, respectively). Unexpectedly, we identified the emergence of a novel BCoV variant characterized by a 12-nt bovine gene insertion in the receptor-binding domain in a natural recombinant HE gene, suggesting a novel evolutionary pattern in BCoV. Electronic supplementary material The online version of this article (10.1007/s00705-020-04840-y) contains supplementary material, which is available to authorized users.

The pandemic outbreak of the Corona viral infection has become a critical global health issue. Biophysical and structural evidence shows that spike protein possesses a high binding affinity towards host angiotensin-converting enzyme 2 and viral hemagglutinin-acetylesterase (HE) glycoprotein receptor. We selected HE as a target in this study to identify potential inhibitors using a combination of various computational approaches such as molecular docking, ADMET analysis, dynamics simulations and binding free energy calculations. Virtual screening of NPACT compounds identified 3,4,5-Trihydroxy-1,8-bis[(2R,3R)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl]benzo[7]annulen-6-one, Silymarin, Withanolide D, Spirosolane and Oridonin as potential HE inhibitors with better binding energy. Furthermore, molecular dynamics simulations for 100 ns time scale revealed that most of the key HE contacts were retained throughout the simulations trajectories. Binding free energy calculations using MM/PBSA approach ranked the top-five potential NPACT compounds which can act as effective HE inhibitors. Graphic abstract Electronic supplementary material The online version of this article (10.1007/s11030-020-10135-w) contains supplementary material, which is available to authorized users.

The role of vascular occlusion in the morbidities, pulmonary and systemic, of COVID-19 has received increasing focus. Histological studies of lung tissue from COVID-19 patients have found extensively damaged endothelium of capillaries adjoining relatively intact alveoli, corresponding to hypoxemia accompanying normal breathing mechanics in such patients. Advanced image analysis of lung CT scans of COVID-19 patients reveals redistribution of blood flow from smaller to larger diameter blood vessels, this effect correlated with the degree of breathing dysfunction. Essential to the study of vascular occlusion in COVID-19 are viral properties dating back to studies of Jonas Salk in the 1940s that have been positively established for SARS-CoV-2. First, SARS-CoV-2 binds to red blood cells (RBCs), in vitro and also clinically in COVID-19 patients. Second, although fusion and replication of SARS-CoV-2 occur via ACE2, such hemagglutinating viruses initially attach to infective targets and clump with blood cells via much more abundantly distributed sialic acid (SA) glycoconjugate binding sites. SARS-CoV-2, in particular, attaches to these SA sites. Third, certain enveloped viruses express an enzyme, hemagglutinin esterase (HE), that counteracts viral-RBC clumping. Notably, among betacoronaviruses, the common cold strains express HE while SARS-CoV-2, SARS-CoV-1 and MERS, the virulent strains, do not. These hemagglutinating properties of SARS-COV-2 establish a framework for “catch and clump” induction of microvascular occlusion proposed here. Ultramicroscopic studies of tissues from COVID-19 patients indicate a key role for hemagglutination early and mid-course in COVID-19, before such clumps harden into clots via the coagulation cascade. Hemagglutination may be reversed by two anti-COVID-19 therapeutics that each competitively bind to SARS-CoV-2 spike protein, blocking such viral attachments. One therapeutic is antiviral antibodies generated by vaccines, the anti-hemagglutination effect of which is exhibited in Jonas Salk’s hemagglutination inhibition assay. The other therapeutic is ivermectin (IVM), a drug of Nobel Prize honored distinction, distributed in 3.7 billion doses worldwide. In ten clinical trials, three with randomized controls, IVM yielded mortality reductions for COVID-19 of 90% at highest doses. IVM may limit virulence of SARS-CoV-2 by steric interference with multivalent spike protein attachments to SA binding sites, blocking hemagglutination, an effect likely to target mutant viral strains.

Influenza C virus is a pathogen that causes acute respiratory illness in children and results in the hospitalization of infants. The antigenicity of the hemagglutinin esterase (HE) glycoprotein is highly stable, and it is not yet known whether antigenic changes contribute to the worldwide transmission and the occurrence of outbreaks of influenza C virus. Here, we performed antigenic analysis of 84 influenza C viruses isolated in Yamagata, Japan, during a 4-year period from 2015 to 2018 and analyzed sequence data for strains of the virus from Japan and many other parts of the world. Antigenic and phylogenetic analyses revealed that 83 strains belonged to the C/Sao Paulo lineage, and two sublineage strains, the Aichi99 sublineage and Victoria2012 sublineage, cocirculated between 2016 and 2018. Aichi99 sublineage strains exhibiting decreased reactivity with the monoclonal antibody YA3 became predominant after 2016, and these strains possessed the K190N mutation. Residue 190 is located in the 190-loop on the top side of the HE protein within a region that is known to show variation that does not impair the biological activity of the protein. The Aichi99 sublineage strains possessing the K190N mutation were detected after 2012 in Europe, Australia, the USA, and Asia as well as Japan. These observations suggest that antigenic variants with K190N mutations have circulated extensively around the world and caused outbreaks in Japan between 2016 and 2018. Our study indicated that the 190-loop is an important antigenic region, and the results suggested that changes in the 190-loop have contributed to the extensive transmission of the virus.

In a published review entitled "COVID-19 diagnosis -A review of current methods", the authors considered hemagglutinin esterase as one of the structural proteins of SARS-CoV-2 and also they did not represent ORF3b, ORF9b, and ORF9c in SARS-CoV-2 genome structure. However, according to the scientific evidence, among coronaviruses only some betacoronaviruses (Embecovirus subgenera) contain HE, and the genome of most of the coronaviruses such as SARS-CoV-2, SARS-CoV, and MERS-CoV lack the HE gene. In addition, the genome of SARS-CoV-2 contains several accessory proteins ORFs including ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10.

Aim: S16, a monoclonal antibody against the hemagglutinin-esterase fusion (HEF) glycoprotein of influenza C virus, reacts with SV40 large T antigen (LT) and a host cellular component(s). The aim is to determine the location of S16 linear epitope on LT and the amino acid sequence of S16 epitope. Materials & methods: BHK-21 cells expressing wild-type and mutant LTs, HEFs or GFPs, each of which was tagged with a FLAG epitope, were analyzed by immunoblotting using S16. Results & conclusions: An amino acid sequence 98-FNEENL-103 on LT forms a linear epitope recognized by S16. The sequence of S16 epitope was defined as F[NAT]EE[NYA]L, excluding FAEEAL and FTEEAL. This finding will be of help in identifying a host cellular component(s) crossreactive with S16.

Genetic recombination is often demonstrated in coronaviruses and can result in host range expansion or alteration in tissue tropism. Here, we showed interspecies events of recombination of an endemic dromedary camel coronavirus, HKU23, with other clade A betacoronaviruses. Our results supported the possibility that the zoonotic pathogen MERS-CoV, which also cocirculates in the same camel species, may have undergone similar recombination events facilitating its emergence or may do so in its future evolution., Genetic recombination has frequently been observed in coronaviruses. Here, we sequenced multiple complete genomes of dromedary camel coronavirus HKU23 (DcCoV-HKU23) from Nigeria, Morocco, and Ethiopia and identified several genomic positions indicative of cross-species virus recombination events among other betacoronaviruses of the subgenus Embecovirus (clade A beta-CoVs). Recombinant fragments of a rabbit coronavirus (RbCoV-HKU14) were identified at the hemagglutinin esterase gene position. Homolog fragments of a rodent CoV were also observed at 8.9-kDa open reading frame 4a at the 3′ end of the spike gene. The patterns of recombination differed geographically across the African region, highlighting a mosaic structure of DcCoV-HKU23 genomes circulating in dromedaries. Our results highlighted active recombination of coronaviruses circulating in dromedaries and are also relevant to the emergence and evolution of other betacoronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV). IMPORTANCE Genetic recombination is often demonstrated in coronaviruses and can result in host range expansion or alteration in tissue tropism. Here, we showed interspecies events of recombination of an endemic dromedary camel coronavirus, HKU23, with other clade A betacoronaviruses. Our results supported the possibility that the zoonotic pathogen MERS-CoV, which also cocirculates in the same camel species, may have undergone similar recombination events facilitating its emergence or may do so in its future evolution.

Introduction Recent data on influenza C virus indicate a possible higher clinical impact in specified patient populations than previously thought. Aim We aimed to investigate influenza C virus circulation in Germany. Methods A total of 1,588 samples from 0 to 4 year-old children presenting as outpatients with influenza-like illness (ILI) or acute respiratory infection were analysed retrospectively. The samples represented a subset of all samples from the German national surveillance system for influenza in this age group in 2012–14. The presence of influenza C virus was investigated by real-time PCR. For positive samples, information on symptoms as well as other respiratory virus co-infections was considered. Retrieved influenza C viral sequences were phylogenetically characterised. Results Influenza C viral RNA was detected in 20 (1.3% of) samples, including 16 during the 2012/13 season. The majority (18/20) of influenza C-positive patients had ILI according to the European Union definition, one patient had pneumonia. Viruses belonged to the C/Sao Paulo and C/Kanagawa lineages. Most (11/20) samples were co-infected with other respiratory viruses. Conclusion Our data are the first on influenza C virus circulation in Germany and notably from a European national surveillance system. The low detection frequency and the identified virus variants confirm earlier observations outside a surveillance system. More virus detections during the 2012/13 season indicate a variable circulation intensity in the different years studied. Influenza C virus can be considered for ILI patients. Future studies addressing its clinical impact, especially in patients with severe disease are needed.

9-O-acetylation of sialic acid is a common modification that plays important roles in host-pathogen interactions. CASD1 has been described as a sialate-O-acetyltransferase and has been shown to be essential for 9-O-acetylation of sialic acid in some cell lines in vitro. In this study, we used knockout mice to confirm that CASD1 is indeed responsible for 9-O-acetylation of sialic acids in vivo. We observed a complete loss of 9-O-acetylation of sialic acids on the surface of myeloid, erythroid and CD4+ T cells in Casd1-deficient mice. Although 9-O-acetylation of sialic acids on multiple hematopoietic lineages was lost, there were no obvious defects in hematopoiesis. Interestingly, red blood cells from Casd1-deficient mice also lost reactivity to TER-119, a rat monoclonal antibody that is widely used to mark the murine erythroid lineage. The sialic acid glyco-epitope recognized by TER-119 on red blood cells was sensitive to the sialic acid O-acetyl esterase activity of the hemagglutinin esterase from bovine coronavirus but not to the corresponding enzyme from the influenza C virus. During erythrocyte development TER-119+ Ery-A and Ery-B cells could be stained by catalytically inactive bovine coronavirus hemaggutinin-esterase but not by the inactive influenza C hemagglutinin esterase, while TER-119+ Ery-C stage cells and mature erythrocytes were recognized by both virolectins. These results suggest that throughout murine erythrocyte development, cells of the erythroid lineage express a glycoconjugate bearing a modified 7,9-di-O-acetyl form of sialic acid, that is recognized specifically by the bovine coronavirus lectin and not by the influenza C hemagglutinin, and this modified sialic acid moiety is a component of the TER-119 epitope. As erythrocytes mature, the surface of Ery-C cells and mature erythrocytes also acquires a distinct CASD1-dependent 9-O-acetyl sialic acid moiety that can be recognized by virolectins from both influenza C and bovine coronavirus that are specific for 9-O-acetyl sialic acid.

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable, however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability.

Coronavirus (CoV), which causes animal diseases, has become a human health concern. Prior to 2003, CoV caused respiratory diseases and enteric disorders, but after 2003, CoV caused three acute respiratory syndromes, resulting in significant human deaths. Since then, research on CoV has multiplied, leading to a deeper knowledge of the species. It is in this context that this article reviews the history, the biological aspect, the evolution and the crossing of the CoV species barrier. This review shows that CoVs are formed by a large genome (27 to 33 kb) and by structural proteins (spike S, hemagglutinin esterase HE and membran protein M). Various coronaviruses have been described in a wide range of species including chickens (IBV-CoV), pigs (PHE-CoV, PED-CoV, TGE-CoV PR-CoV, PD-CoV, SADS-CoV), cattle (BCoV), cats (FCoV), dogs (CCoV), and humans (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV2). Birds and bats are the main reservoirs of CoVs, but due to the low fidelity of the replication complex, CoVs have the ability to adapt to various species. Due to the crossing of the species barrier, CoVs have a wide host range resulting in the emergence of various strains worldwide. This information can help researchers develop intervention strategies to prevent the re-emergence of CoVs in the future.

In an earlier study on respiratory infections in Singapore military recruits, four influenza C virus (FLUCV) infections were detected out of the 1354 samples collected. All four isolates were detected in 2006, and their whole genome was completely sequenced and analysed. Phylogenetic analysis of the hemagglutinin esterase fusion (HEF) gene revealed that all four Singapore isolates belonged to the C/Japan-Kanagawa/1/76-related lineage. However, the genes of the four FLUCV isolates had origins from several different lineages, and the genome composition resembles that of the C/Japan-Miyagi/9/96-like strains that had been circulating in Japan between 1996 and 2000.

The 2019-novel coronavirus (nCoV) is a major source of disaster in the 21th century. However, the lack of specific drugs to prevent/treat an attack is a major need at this current point of time. In this regard, we conducted a systematic review to identify major druggable targets in coronavirus (CoV). We searched PubMed and RCSB database with keywords HCoV, NCoV, corona virus, SERS-CoV, MERS-CoV, 2019-nCoV, crystal structure, X-ray crystallography structure, NMR structure, target, and drug target till Feb 3, 2020. The search identified seven major targets (spike protein, envelop protein, membrane protein, protease, nucleocapsid protein, hemagglutinin esterase, and helicase) for which drug design can be considered. There are other 16 nonstructural proteins (NSPs), which can also be considered from the drug design perspective. The major structural proteins and NSPs may serve an important role from drug design perspectives. However, the occurrence of frequent recombination events is a major deterrent factor toward the development of CoV-specific vaccines/drugs.

We mapped the hemagglutinin-esterase (HE) antigenic epitopes of the influenza C virus on the three-dimensional (3D) structure of the HE glycoprotein using 246 escape mutants that were selected by a panel of nine anti-HE monoclonal antibodies (MAbs), including seven of the C/Ann Arbor/1/50 virus and two of the C/Yamagata/15/2004 virus. The frequency of variant selection in the presence of anti-HE MAbs was very low, with frequencies ranging from 10&minus, 4.62 to 10&minus, 7.58 for the C/Ann Arbor/1/50 virus and from 10&minus, 7.11 to 10&minus, 9.25 for the C/Yamagata/15/2004 virus. Sequencing of mutant HE genes revealed 25 amino acid substitutions at 16 positions in three antigenic sites: A-1, A-2, and A-3, and a newly designated Y-1 site. In the 3D structure, the A-1 site was widely located around the receptor-binding site, the A-2 site was near the receptor-destroying enzyme site, and the Y-1 site was located in the loop on the topside of HE. The hemagglutination inhibition reactions of the MAbs with influenza C viruses, circulating between 1947 and 2016, were consistent with the antigenic-site amino acid changes. We also found some amino acid variations in the antigenic site of recently circulating strains with antigenic changes, suggesting that viruses that have the potential to alter antigenicity continue to circulate in humans.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax ( n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 10 5 50% tissue culture infective dose(s) (TCID 50 )] D/OK ( n = 6) or D/600 ( n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine ( n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 10 5 TCID 50 ) with IDV D/OK ( n = 6) or D/660 ( n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model. IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.

The only spike of influenza C virus, the hemagglutinin-esterase-fusion glycoprotein (HEF) combines receptor binding, receptor hydrolysis and membrane fusion activities. Like other hemagglutinating glycoproteins of influenza viruses HEF is S-acylated, but only with stearic acid at a single cysteine located at the cytosol-facing end of the transmembrane region. Previous studies established the essential role of S-acylation of hemagglutinin for replication of influenza A and B virus by affecting budding and/or membrane fusion, but the function of acylation of HEF was hitherto not investigated. Using reverse genetics we rescued a virus containing non-stearoylated HEF, which was stable during serial passage and showed no competitive fitness defect, but the growth rate of the mutant virus was reduced by one log. Deacylation of HEF does neither affect the kinetics of its plasma membrane transport nor the protein composition of virus particles. Cryo-electron microscopy showed that the shape of viral particles and the hexagonal array of spikes typical for influenza C virus were not influenced by this mutation indicating that virus budding was not disturbed. However, the extent and kinetics of haemolysis were reduced in mutant virus at 37°C, but not at 33°C, the optimal temperature for virus growth, suggesting that non-acylated HEF has a defect in membrane fusion under suboptimal conditions.

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9- O -acetylated sialic acid (9- O -Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9- O -Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate- O -acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O -acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O -Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate- O -acetylesterase RDE activity. IMPORTANCE Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O -acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O -acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate- O -acetylesterase RDE activity.

Influenza D virus (IDV) utilizes cattle as a primary reservoir. Increased outbreaks in pigs and serological evidence of human infection have raised a concern about the potential of IDV adapting to humans. Here, we directly compared IDV’s stability to that of other influenza types (A, B, and C) following prolonged incubation at high temperatures or in a low-pH environment. We found that IDV is the most stable of the four types of influenza viruses. Importantly, we demonstrated that the hemagglutinin-esterase fusion (HEF) protein, which drives the fusion between viral and host cell membranes, is the primary determinant for the high thermal and acid stability of IDV. Considering that there is a link between the acid stability of the hemagglutinin protein of influenza A virus and its cross-species transmission, further investigation of the mechanism of HEF-directed viral tolerance may offer novel insights into tissue tropism and cross-species transmission of influenza viruses., Influenza D virus (IDV) is unique among four types of influenza viruses in that it utilizes cattle as a primary reservoir. The thermal and acid stability of IDV were examined and directly compared with those of influenza A virus (IAV), influenza B virus (IBV), and influenza C virus (ICV). The results of our experiments demonstrated that only IDV had a high residual infectivity (~2.5 log units of 50% tissue culture infective dose [TCID50]/ml) after a 60-min exposure to 53°C in solution at a neutral pH, and remarkably, IDV retained this infectivity even after exposure to 53°C for 120 min. Furthermore, the data showed that IDV was extremely resistant to inactivation by low pH. After being treated at pH 3.0 for 30 min, IDV lost only approximately 20% of its original infectiousness, while all other types of influenza viruses were completely inactivated. Finally, replacement of the hemagglutinin (HA) and neuraminidase (NA) proteins of a temperature- and acid-sensitive IAV with the hemagglutinin-esterase fusion (HEF) protein of a stable IDV through a reverse genetic system largely rendered the recombinant IAVs resistant to high-temperature and low-pH treatments. Together, these results indicated that the HEF glycoprotein is a primary determinant of the exceptional temperature and acid tolerance of IDV. Further investigation into the viral entry and fusion mechanism mediated by the intrinsically stable HEF protein of IDV may offer novel insights into how the fusion machinery of influenza viruses evolve to achieve acid and thermal stability, which as a result promotes the potential to transmit across mammal species. IMPORTANCE Influenza D virus (IDV) utilizes cattle as a primary reservoir. Increased outbreaks in pigs and serological evidence of human infection have raised a concern about the potential of IDV adapting to humans. Here, we directly compared IDV’s stability to that of other influenza types (A, B, and C) following prolonged incubation at high temperatures or in a low-pH environment. We found that IDV is the most stable of the four types of influenza viruses. Importantly, we demonstrated that the hemagglutinin-esterase fusion (HEF) protein, which drives the fusion between viral and host cell membranes, is the primary determinant for the high thermal and acid stability of IDV. Considering that there is a link between the acid stability of the hemagglutinin protein of influenza A virus and its cross-species transmission, further investigation of the mechanism of HEF-directed viral tolerance may offer novel insights into tissue tropism and cross-species transmission of influenza viruses.

Background Infectious salmon anemia virus (ISAV) is an important fish pathogen that causes high mortality in farmed Atlantic salmon. The ISAV genome consists of eight single-stranded, negative-sense RNA segments. The six largest segments contain one open reading frame (ORF) each, and encode three polymerase proteins, nucleoprotein, fusion protein, and hemagglutinin esterase protein. The two smallest segments contain more than one ORF each. The segment 7 encodes non-structural protein 1 (NS1) and nuclear export protein (NEP), while segment 8 encodes matrix protein 1 and 2 (M1 and M2). NS1 and M2 have been well known as antagonist of type I interferon. However, little is known about the characterization of M1 or NEP. In addition, heat shock cognate 70 (Hsc70) has been reported to interact with M1 and NEP of influenza viruses for the export of viral ribonucleoprotein (vRNP) via vRNP-M1-NEP complex, the goal of this study therefore was to characterize the subcellular localization and interactions of ISAV M1 and NEP as well as cellular Hsc70. Results When M1, NEP, and Hsc70 were individually expressed in the stripped snakehead (SSN-1) cells, we found that M1 protein was localized in both cytosol and nucleus of the cells, NEP was localized only in the cytosol and accumulated adjacent to the nucleus, while Hsc70 was localized throughout the cytosol, but not in the nucleus. However, when two of them were co-expressed, we found that both M1 and Hsc70 were co-localized with NEP in the cytosol and accumulated adjacent to the nucleus, while M1 and Hsc70 were still localized as they were expressed individually. Furthermore, pull-down assay was performed and showed that NEP could interact with both M1 and Hsc70, and M1-Hsc70 interaction was also observed although the interaction was weaker than that of NEP-Hsc70. Conclusion Our study characterized the subcellular localization and interactions of three proteins including M1 and NEP of ISAV, and Hsc70. These data will help towards a better understanding of the life cycle of ISAV, especially the process of vRNP export.

Viruses with approximately 50% homology to human influenza C virus (ICV) have recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody cross-reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays, and inability to productively reassort with ICV led to the proposal that these viruses represented a new genus of influenza virus, influenzavirus D (IDV). To further our understanding of the epidemiology of IDV, real-time reverse transcription-PCR was performed on a set of 208 samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six viruses were successfully isolatedin vitro. Phylogenetic analysis of full-genome sequences of these six new viruses and four previously reported viruses revealed two distinct cocirculating lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660), which frequently reassorted with one another. Antigenic analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an approximate 10-fold loss in cross-reactivity against heterologous clade antiserum. One isolate, D/bovine/Texas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic determinant responsible for the discrepant HI results. These results suggest that IDV is common in bovines with respiratory disease and that at least two genetic and antigenically distinct clades cocirculate.IMPORTANCEA novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV). Here, we show that IDV is common in clinical samples of bovine respiratory disease complex (BRDC), with a prevalence similar to that of other established BRDC etiological agents. These results are in good agreement with the near-ubiquitous seroprevalence of IDV previously found. Phylogenetic analysis of complete genome sequences found evidence for two distinct cocirculating lineages of IDV which freely reassort. Significant antigenic differences, which generally agreed with the surface glycoprotein hemagglutinin esterase phylogeny, were observed between the two lineages. Based on these results, and on the ability of IDV to infect and transmit in multiple mammalian species, additional studies to determine the pathogenic potential of IDV are warranted.

The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin–esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection. Electronic supplementary material The online version of this article (doi:10.1007/s11262-013-0926-y) contains supplementary material, which is available to authorized users.

Most betacoronaviruses possess an hemagglutinin-esterase (HE) protein, which appears to play a role in binding to or release from the target cell. Since this HE protein possesses an acetyl-esterase activity that removes acetyl groups from O-acetylated sialic acid, a role as a receptor-destroying enzyme has been postulated. However, the precise function of HE and of its enzymatic activity remains poorly understood. Making use of neutralizing antibody and of molecular clones of recombinant human coronavirus OC43 (HCoV-OC43), our results suggest that the HE protein of this HCoV could be associated with infection of target cells and, most notably, is important in the production of infectious viral particles. Indeed, after transfecting BHK-21 cells with various cDNA infectious clones of HCoV-OC43, either lacking the HE protein or bearing an HE protein with a nonfunctional acetyl-esterase enzymatic activity, we were reproducibly unable to detect recombinant infectious viruses compared to the reference infectious HCoV-OC43 clone pBAC-OC43 FL . Complementation experiments, using BHK-21 cells expressing wild-type HE, either transiently or in a stable ectopic expression, demonstrate that this protein plays a very significant role in the production of infectious recombinant coronaviral particles that can subsequently more efficiently infect susceptible epithelial and neuronal cells. Even though the S protein is the main viral factor influencing coronavirus infection of susceptible cells, our results taken together indicate that a functionally active HE protein enhances the infectious properties of HCoV-OC43 and contributes to efficient virus dissemination in cell culture.

A replicon expression system based on the salmonid alphavirus (SAV) that encodes the infectious salmon anemia virus (ISAV) hemagglutinin-esterase (HE) was constructed and found to be an efficacious vaccine against infectious salmon anemia (ISA). Following a single intramuscular immunization, Atlantic salmon (Salmo salar) were effectively protected against subsequent ISAV challenge. Additional replicons coding for the ISAV fusion glycoprotein (F) or the ISAV matrix protein (M) were created and tested in combination with the replicon that encodes the HE. The ISAV HE was confirmed as a potent antigen, but neither the F nor the M proteins were found to be essential for immunization-induced protection. Innate immune response induced at the site of vaccination illustrated the immunogenicity of the SAV-based replicon and its ability to activate antiviral responses in Atlantic salmon. The successful testing of the SAV-based replicon as a vaccine model against ISA showed that the replicon approach may represent a novel immunization technology for the aquaculture industry. It offers potential benefits in terms of safety, efficacy, flexibility, and vaccine production complexity.

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1) multiple lineages have been circulating globally; (2) there have been weak and infrequent selective bottlenecks; (3) the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4) there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics.

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein-carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.

Plasmids expressing interferon (IFN) have recently been shown to function as adjuvants in Atlantic salmon when co-injected with a DNA vaccine encoding hemagglutinin-esterase (HE) from infectious salmon anemia virus (ISAV). In this work we have compared the antibody kinetics and the systemic Mx/ISG15 response of fish vaccinated with HE-plasmid using either IFNa plasmid (pIFNa) or pIFNc as adjuvants over a longer time period, i.e. 22 weeks post vaccination (pv). The results showed that the antibody response against ISAV with pIFNa as adjuvant arose earlier (7 weeks pv) than with pIFNc as adjuvant (10 weeks pv), peaked at week 10 and declined at week 22. The antibody response with pIFNc as adjuvant peaked at 16 weeks and kept at this level 22 weeks pv. Fish injected with pIFNc alone expressed high levels of Mx and ISG15 in liver throughout the 22 week period. In contrast, fish injected with pIFNc together with HE-plasmid expressed high levels of Mx and ISG15 in liver for the first 10 weeks, but at week 16 this response was absent in two of three fish at week 16 and was absent in all tested fish at week 22 pv. This suggests that cells expressing HE and IFNc are intact at week 10 pv, but are eliminated by adaptive immune responses after week 10 due to recognition of HE. The longevity of the Mx/ISG15 response in pIFNc treated fish is likely due to the fact that IFNc is a self-antigen of salmon and is not attacked by the adaptive immune system.