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.

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.

Glycan remodeled erythrocytes facilitate antigenic characterization of recent A/H3N2 influenza viruses.

During circulation in humans and natural selection to escape antibody recognition for decades, A/H3N2 influenza viruses emerged with altered receptor specificities. These viruses lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Examination of receptor specificities of A/H3N2 viruses reveals that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing α2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics shows an absence of extended glycans providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installing functional receptors on erythrocytes, allows antigenic characterization of recent A/H3N2 viruses confirming the cocirculation of antigenically different viruses in humans. Computational analysis of HAs in complex with sialosides having extended LacNAc moieties reveals that mutations distal to the RBD reoriented the Y159 side chain resulting in an extended receptor binding site.

Reversible O-Acetyl Migration within the Sialic Acid Side Chain and Its Influence on Protein Recognition.

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-O-acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-O-acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host-microbe interactions and potential application of the more stable synthetic N-acetyl mimics.

In situ structure and organization of the influenza C virus surface glycoprotein.

The lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF's membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo.

To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

A protein folding robot driven by a self-taught agent.

This paper presents a computer simulation of a virtual robot that behaves as a peptide chain of the Hemagglutinin-Esterase protein (HEs) from human coronavirus. The robot can learn efficient protein folding policies by itself and then use them to solve HEs folding episodes. The proposed robotic unfolded structure inhabits a dynamic environment and is driven by a self-taught neural agent. The neural agent can read sensors and control the angles and interactions between individual amino acids. During the training phase, the agent uses reinforcement learning to explore new folding forms that conduce toward more significant rewards. The memory of the agent is implemented with neural networks. These neural networks are noise-balanced trained to satisfy the look for future conditions required by the Bellman equation. In the operating phase, the components merge into a wise up protein folding robot with look-ahead capacities, which consistently solves a section of the HEs protein.

Structure based virtual screening identifies small molecule effectors for the sialoglycan binding protein Hsa.

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialylated glycans displayed on human platelets. Despite their important role in disease progression, there are currently no anti-adhesive drugs available on the market. Here, we performed structure-based virtual screening using an ensemble docking approach followed by consensus scoring to identify novel small molecule effectors against the sialoglycan binding domain of the SRR adhesin protein Hsa from the S. gordonii strain DL1. The screening successfully predicted nine compounds which were able to displace the native ligand (sialyl-T antigen) in an in vitro assay and bind competitively to Hsa. Furthermore, hierarchical clustering based on the MACCS fingerprints showed that eight of these small molecules do not share a common scaffold with the native ligand. This study indicates that SRR family of adhesin proteins can be inhibited by diverse small molecules and thus prevent the interaction of the protein with the sialoglycans. This opens new avenues for discovering potential drugs against IE.

Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans.

The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles contain two surface projections called spike (S) and haemagglutinin-esterase (HE), with S mediating receptor binding and membrane fusion, and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors, specifically 9-O-acetylated sialic acid. Here we present the cryo-EM structure of the ~80 kDa, heavily glycosylated HKU1 HE at 3.4 Å resolution. Comparison with existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the esterase domain left intact. Cryo-EM and mass spectrometry analysis reveals a putative glycan shield on the now redundant lectin domain. The findings further our insight into the evolution and host adaptation of human embecoviruses, and demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins.

Microcavity array supported lipid bilayer models of ganglioside - influenza hemagglutinin1 binding.

The binding of influenza receptor (HA1) to membranes containing different glycosphingolipid receptors was investigated at Microcavity Supported Lipid Bilayers (MSLBs). We observed that HA1 preferentially binds to GD1a but the diffusion coefficient of the associated complex at lipid bilayer is approximately double that of the complexes formed by HA1 GM1 or GM3.

Molecular Characterization of Influenza C Viruses from Outbreaks in Hong Kong SAR, China.

In 2014, the Centre for Health Protection in Hong Kong introduced screening for influenza C virus (ICV) as part of its routine surveillance for infectious agents in specimens collected from patients presenting with symptoms of respiratory viral infection, including influenza-like illness (ILI). A retrospective analysis of ICV detections up to week 26 of 2019 revealed persistent low-level circulation, with two outbreaks having occurred in the winters of 2015 to 2016 and 2017 to 2018. These outbreaks occurred at the same time as, and were dwarfed by, seasonal epidemics of influenza types A and B. Gene sequencing studies on stored ICV-positive clinical specimens from the two outbreaks have shown that the hemagglutinin-esterase (HE) genes of the viruses fall into two of the six recognized genetic lineages (represented by C/Kanagawa/1/76 and C/São Paulo/378/82), with there being significant genetic drift compared to earlier circulating viruses within both lineages. The location of a number of encoded amino acid substitutions in hemagglutinin-esterase fusion (HEF) glycoproteins suggests that antigenic drift may also have occurred. Observations of ICV outbreaks in other countries, with some of the infections being associated with severe disease, indicates that ICV infection has the potential to have significant clinical and health care impacts in humans.IMPORTANCE Influenza C virus infection of humans is common, and reinfection can occur throughout life. While symptoms are generally mild, severe disease cases have been reported, but knowledge of the virus is limited, as little systematic surveillance for influenza C virus is conducted and the virus cannot be studied by classical virologic methods because it cannot be readily isolated in laboratories. A combination of systematic surveillance in Hong Kong SAR, China, and new gene sequencing methods has been used in this study to assess influenza C virus evolution and provides evidence for a 2-year cycle of disease outbreaks. The results of studies like that reported here are key to developing an understanding of the impact of influenza C virus infection in humans and how virus evolution might be associated with epidemics.

Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential.

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.

Triple combination therapy of favipiravir plus two monoclonal antibodies eradicates influenza virus from nude mice.

Prolonged treatment of immunocompromised influenza patients with viral neuraminidase (NA) inhibitors is required, because the immune system of such patients fails to eradicate the viruses. Here, we attempted to eradicate influenza virus from the respiratory organs of nude mice, which is a model of immunocompromised hosts, by using combination therapy of the viral polymerase inhibitor favipiravir and monoclonal antibodies (mAbs) against the receptor-binding site (RBS) and stem of viral hemagglutinin (HA). Although monotherapy or combination therapy of two antivirals (two mAbs or favipiravir plus a mAb) suppressed virus replication, they failed to eradicate viruses from nude mice. In contrast, the triple combination therapy of favipiravir plus anti-Stem and anti-RBS mAbs completely stopped virus replication in nude mice, resulting in virus clearance. Triple combination approaches should be considered for the treatment of human immunocompromised patients with severe influenza.

Structural characteristics of measles virus entry.

Measles virus, a member of the genus Morbillivirus, is highly contagious and still shows considerable mortality with over 100000 deaths annually, although efficient attenuated vaccines exist. Recent studies of measles virus haemagglutinin (MeV-H) and its receptor, including crystallographic and electron microscopic structural analyses combined with functional assays, have revealed how the MeV-H protein recognizes its cognate receptors, SLAM and Nectin-4, and how the glycan shield ensures effective vaccination. In addition, the crystal structure of the MeV-F protein indicated its similarity to those of other paramyxoviruses. Taking into account these data, several models of viral entry/membrane fusion of measles viruses and related paramyxoviruses have been proposed. Furthermore, anti-MeV-F inhibitors targeted to specific regions to inhibit MeV-F protein activation were reported, with potency for preventing MeV infection. The inhibitors targeted for entry events may potentially be applied to treatment of MeV-derived diseases, although escape mutations and drug profiles should be considered.

Hemagglutinin Inhibitors are Potential Future Anti-Influenza Drugs for Mono- and Combination Therapies.

Infections by H1-H16 influenza A viruses require sufficient binding of viral hemagglutinins (HAs) to specific target receptors, glycoconjugates bearing sialyl sugar chains, on the host cell surface. Synthesized sialyl sugar chains targeting sialyl sugar-binding sites in HAs that are immutable as long as the virus does not switch to a different host species might therefore be highly effective candidate drugs for inhibition of the initial required step of virus entry. In this chapter, we describe the following aspects of updated sialyl sugar chains as influenza A virus HA inhibitors (HAIs): (1) mode of terminal sialyl-galactose linkage, (2) molecular length and structure of sialyl glycan receptors, (3) multivalent sialyl sugar chain dimension, (4) clustering of sialyl sugar chains on macromolecular scaffolds, and (5) enhancement of the stability of sialyl sugar chain HA inhibitors. We also discuss about the use of HAI-based combinations that should be considered for future influenza therapy.

Optimization of 4-Aminopiperidines as Inhibitors of Influenza A Viral Entry That Are Synergistic with Oseltamivir.

Vaccination is the most prevalent prophylactic means for controlling seasonal influenza infections. However, an effective vaccine usually takes at least 6 months to develop for the circulating strains. Therefore, new therapeutic options are needed for the acute treatment of influenza infections to control this virus and prevent epidemics/pandemics from developing. We have discovered fast-acting, orally bioavailable acylated 4-aminopiperidines with an effective mechanism of action targeting viral hemagglutinin (HA). Our data show that these compounds are potent entry inhibitors of influenza A viruses. We present docking studies that suggest an HA binding site for these inhibitors on H5N1. Compound 16 displayed a significant decrease of viral titer when evaluated in the infectious assays with influenza virus H1N1 (A/Puerto Rico/8/1934) or H5N1 (A/Vietnam/1203/2004) strains and the oseltamivir-resistant strain with the most common H274Y mutation. In addition, compound 16 showed significant synergistic activity with oseltamivir in vitro.

Identification of antigenic epitopes in the haemagglutinin protein of H7 avian influenza virus.

The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The haemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs, 2F8, 3F6, 5C11, 5E2 and 5C12) against the HA protein of H7 virus were produced and characterized. Epitope mapping indicated that 103RESGSS107 was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and mAbs 5E2/5C12 recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and none of the five mAbs cross-reacted with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope 103RESGSS107 was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations prevented the mutated epitope from being recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that 103RESGSS107 may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus.RESEARCH HIGHLIGHTSFive mAbs against HA protein of H7 AIV were generated and characterized.Two novel epitopes 103RESGSS107 and 103-145aa were identified.The epitope 103RESGSS107 differs between Eurasian and North-American lineages.The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.

Phylodynamic analysis of two amino acid substitutions in the hemagglutinin protein of canine distemper virus strains detected in fur-bearing animals in China.

Canine distemper virus (CDV) causes a highly contagious disease in a wide range of carnivores. The hemagglutinin (H) protein of viruses shows the highest variability and plays an important role in modulation of viral antigenicity, virulence, and receptor recognition. Since 2012, canine distemper (CD) outbreaks in fur-bearing animals (minks, foxes, raccoon dogs) caused by CDV variants with I542N and Y549H substitutions in the H protein have been frequently reported in China. To characterize the molecular evolutionary dynamics and epidemiological dynamics of CDV, 235 H gene sequences of CDV wild-type strains collected from 22 countries between 1975 and 2015, including 44 strains predominant in fur-bearing animals in China, were analyzed. The phylogenetic relationships and evolutionary rates of the CDV strains were determined by Bayesian phylogenetics. The CDV strains clustered into distinct geographic genotypes, irrespective of the species of isolation. All the variant strains formed a distinct monophyletic cluster and belonged to the F sub-genotype within the Asia-1 genotype-currently the predominant sub-genotype in fur-bearing animals in China. Evolutionary analysis suggested that the variant strains originated in 2006. Furthermore, the selection pressure analysis revealed that the Y549H substitution was under positive selection pressure for adaptation toward the fur-bearing animals. The residue at position 549 also showed structural interaction with the V domain of the mink signaling lymphocyte-activation molecule (SLAM) receptor based on the homology modeling of the H-SLAM complex. Our results suggested that the Y549H substitution contributed to the molecular adaptation of CDV variants in the fur-bearing animals during the viral evolutionary phase in China.