Hemagglutinin Sequences Research Articles

Molecular characterization and evolution dynamics of influenza B viruses circulating in Germany from season 1996/1997 to 2019/2020

Influenza B viruses are responsible for significant disease burden caused by viruses of both the Yamagata- and Victoria-lineage. Since the circulating patterns of influenza B viruses in different countries vary we investigated molecular properties and evolution dynamics of influenza B viruses circulating in Germany between 1996 and 2020. A change of the dominant lineage occurred in Germany in seven seasons in over past 25 years. A total of 676 sequences of hemagglutinin coding domain 1 (HA1) and 516 sequences of neuraminidase (NA) genes of Yamagata- and Victoria-lineage viruses were analyzed using time-scaled phylogenetic tree. Phylogenetic analysis demonstrated that Yamagata-lineage viruses are more diverse than the Victoria-lineage viruses and could be divided into nine genetic groups whereas Victoria-lineage viruses presented six genetic groups. Comparative phylogenetic analyses of both the HA and NA segments together revealed a number of inter-lineage as well as inter- and intra-clade reassortants. We identified key amino acid substitutions in major HA epitopes such as in four antigenic sites and receptor-binding sites (RBS) and in the regions close to them, with most substitutions in the 120-loop of both lineage viruses. Altogether, seventeen substitutions were fixed over time within the Yamagata-lineage with twelve of them in the antigenic sites. Thirteen substitutions were identified within the Victoria-lineage, with eleven of them in the antigenic sites. Moreover, all Victoria-lineage viruses of the 2017/2018 season were characterized by a deletion of two amino acids at the position 162–163 in the antigenic site of HA1. The viruses with triple deletion Δ162–164 were found in Germany since season 2018/2019. We highlighted the interplay between substitutions in the glycosylation sites and RBS and antigenic epitope during HA evolution. The results obtained underscore the need for continuous monitoring of circulating influenza B viruses. Early detection of strains with genetic and antigenic variation is essential to predict the circulation patterns for the following season. Such information is important for the development of optimal vaccines and strategies for prevention and control of influenza.

Reconstruction of molecular evolution of human influenza A H1N1/2009 virus in Iran and neighboring countries.

In this study, the time and path of transmission of H1N1 serotype influenza A viruses in Iran and neighboring countries have been investigated by using Bayesian phylogeography analysis on the sequences extracted from the gene bank. We obtained all hemagglutinin (HA) and neuraminidase (NA) nucleotide sequences of influenza H1N1 available up to December 25, 2020, from Iran and its neighboring countries (i.e., Pakistan, Afghanistan, Turkmenistan, Armenia, Azerbaijan, Turkey, and Iraq). We also performed a Bayesian Markov chain Monte Carlo method to infer the evolutionary dynamic and the most recent common ancestor for the HA and NA sequences. Based on the extracted sequences, the age of emergence of H1N1 influenza virus serotype was older in Iran compared to neighboring countries, and with some degree of uncertainty, it seems Tehran had a key role and epicenter of transmission to other cities within Iran. The mean time of the most recent common ancestor of H1N1 viruses was 1989 (95% HPD: 1980-1994) for HA and NA as well. Along with ordinary measures like resource management, diagnostic approaches, and preparedness to fight against viruses that were in place, continuous monitoring, and screening of H1N1 serotype influenza virus in the country, especially by implementation of feasible, effective, and innovative measures at borderline should be initiated and identified gaps and shortage that should be a priority for virus control. It is also important for countries to have a regional monitoring program in addition to internal monitoring programs, as well as to start a virus molecular care program.

Dive into machine learning algorithms for influenza virus host prediction with hemagglutinin sequences

Influenza viruses mutate rapidly and can pose a threat to public health, especially to those in vulnerable groups. Throughout history, influenza A viruses have caused pandemics between different species. It is important to identify the origin of a virus in order to prevent the spread of an outbreak. Recently, there has been increasing interest in using machine learning algorithms to provide fast and accurate predictions for viral sequences. In this study, real testing data sets and a variety of evaluation metrics were used to evaluate machine learning algorithms at different taxonomic levels. As hemagglutinin is the major protein in the immune response, only hemagglutinin sequences were used and represented by position-specific scoring matrix and word embedding. The results suggest that the 5-grams-transformer neural network is the most effective algorithm for predicting viral sequence origins, with approximately 99.54% AUCPR, 98.01% F1 score and 96.60% MCC at a higher classification level, and approximately 94.74% AUCPR, 87.41% F1 score and 80.79% MCC at a lower classification level.

Use of the ISU FLUture multisequence identity tool for rapid interpretation of swine influenza A virus sequences in the United States.

Rapid and reliable identification of the hemagglutinin (HA) and neuraminidase (NA) genetic clades of an influenza A virus (IAV) sequence from swine can inform control measures and multivalent vaccine composition. Current approaches to genetically characterize HA or NA sequences are based on nucleotide similarity or phylogenetic analyses. Public databases exist to acquire IAV genetic sequences for comparison, but personnel at the diagnostic or production level have difficulty in adequately updating and maintaining relevant sequence datasets for IAV in swine. Further, phylogenetic analyses are time intensive, and inference drawn from these methods is impacted by input sequence data and associated metadata. We describe here the use of the IAV multisequence identity tool as an integrated public webpage located on the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) FLUture website: https://influenza.cvm.iastate.edu/. The multisequence identity tool uses sequence data derived from IAV-positive cases sequenced at the ISU-VDL, employs a BLAST algorithm that identifies sequences that are genetically similar to submitted query sequences, and presents a tabulation and visualization of the most genetically similar IAV sequence and associated metadata from the FLUture database. Our tool removes bioinformatic barriers and allows clients, veterinarians, and researchers to rapidly classify and identify IAV sequences similar to their own sequences to augment interpretation of results.

Hemagglutinin Subtype Specificity and Mechanisms of Highly Pathogenic Avian Influenza Virus Genesis.

Highly Pathogenic Avian Influenza Viruses (HPAIVs) arise from low pathogenic precursors following spillover from wild waterfowl into poultry populations. The main virulence determinant of HPAIVs is the presence of a multi-basic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein. The MBCS allows for HA cleavage and, consequently, activation by ubiquitous proteases, which results in systemic dissemination in terrestrial poultry. Since 1959, 51 independent MBCS acquisition events have been documented, virtually all in HA from the H5 and H7 subtypes. In the present article, data from natural LPAIV to HPAIV conversions and experimental in vitro and in vivo studies were reviewed in order to compile recent advances in understanding HA cleavage efficiency, protease usage, and MBCS acquisition mechanisms. Finally, recent hypotheses that might explain the unique predisposition of the H5 and H7 HA sequences to obtain an MBCS in nature are discussed.

Characterizing Receptor Flexibility to Predict Mutations That Lead to Human Adaptation of Influenza Hemagglutinin.

A key step in the emergence of human pandemic influenza strains has been a switch in binding preference of the viral glycoprotein hemagglutinin (HA) from avian to human sialic acid (SA) receptors. The conformation of the bound SA varies substantially with HA sequence, and crystallographic evidence suggests that the bound SA is flexible, making it difficult to predict which mutations are responsible for changing HA-binding preference. We performed molecular dynamics (MD) simulations of SA analogues binding to various HAs and observed a dynamic equilibrium among structurally diverse receptor conformations, including conformations that have not been experimentally observed. Using one such novel conformation, we predicted—and experimentally confirmed—a set of mutations that substantially increased an HA’s affinity for a human SA analogue. This prediction could not have been inferred from the existing crystal structures, suggesting that MD-generated HA–SA conformational ensembles could help researchers predict human-adaptive mutations, aiding surveillance of emerging pandemic threats.

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis.

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.

Cross-reactive immunity potentially drives global oscillation and opposed alternation patterns of seasonal influenza A viruses

Several human pathogens exhibit distinct patterns of seasonality and circulate as pairs. For instance, influenza A virus subtypes oscillate and peak during winter seasons of the world’s temperate climate zones. Alternation of dominant strains in successive influenza seasons makes epidemic forecasting a major challenge. From the start of the 2009 influenza pandemic we enrolled influenza A virus infected patients (n = 2980) in a global prospective clinical study. Complete hemagglutinin sequences were obtained from 1078 A/H1N1 and 1033 A/H3N2 viruses. We used phylodynamics to construct high resolution spatio-temporal phylogenetic hemagglutinin trees and estimated global influenza A effective reproductive numbers (R) over time (2009–2013). We demonstrate that R oscillates around R = 1 with a clear opposed alternation pattern between phases of the A/H1N1 and A/H3N2 subtypes. Moreover, we find a similar alternation pattern for the number of global viral spread between the sampled geographical locations. Both observations suggest a between-strain competition for susceptible hosts on a global level. Extrinsic factors that affect person-to-person transmission are a major driver of influenza seasonality. The data presented here indicate that cross-reactive host immunity is also a key intrinsic driver of influenza seasonality, which determines the influenza A virus strain at the onset of each epidemic season.

Mutation in Hemagglutinin Antigenic Sites in Influenza A pH1N1 Viruses from 2015-2019 in the United States Mountain West, Europe, and the Northern Hemisphere.

H1N1 influenza A virus is a respiratory pathogen that undergoes antigenic shift and antigenic drift to improve viral fitness. Tracking the evolutionary trends of H1N1 aids with the current detection and the future response to new viral strains as they emerge. Here, we characterize antigenic drift events observed in the hemagglutinin (HA) sequence of the pandemic H1N1 lineage from 2015–2019. We observed the substitutions S200P, K147N, and P154S, together with other mutations in structural, functional, and/or epitope regions in 2015–2019 HA protein sequences from the Mountain West region of the United States, the larger United States, Europe, and other Northern Hemisphere countries. We reconstructed multiple phylogenetic trees to track the relationships and spread of these mutations and tested for evidence of selection pressure on HA. We found that the prevalence of amino acid substitutions at positions 147, 154, 159, 200, and 233 significantly changed throughout the studied geographical regions between 2015 and 2019. We also found evidence of coevolution among a subset of these amino acid substitutions. The results from this study could be relevant for future epidemiological tracking and vaccine prediction efforts. Similar analyses in the future could identify additional sequence changes that could affect the pathogenicity and/or infectivity of this virus in its human host.

Characterization of Vaccine Breakthrough Cases during Measles Outbreaks in Milan and Surrounding Areas, Italy, 2017-2021.

Despite the existence of an effective live-attenuated vaccine, measles can appear in vaccinated individuals. We investigated breakthrough measles cases identified during our surveillance activities within the measles/rubella surveillance network (MoRoNet) in Milan and surrounding areas (Northern Italy). Between 2017 and 2021, we confirmed measles virus (genotypes B3 or D8) infections in 653 patients and 51 of these (7.8%) were vaccinees. Among vaccinated individuals whose serum was available, a secondary failure was evidenced in 69.4% (25/36) of cases while 11 patients (30.6%) were non-responders. Non-responders were more frequently hospitalized and had significantly lower Ct values in both respiratory and urine samples. Median age and time since the last immunization were similar in the two groups. Importantly, we identified onward transmissions from vaccine failure cases. Vaccinees were involved in 20 outbreaks, in 10 of them they were able to transmit the virus, and in 8 of them, they were the index case. Comparing viral hemagglutinin sequences from vaccinated and non-vaccinated subjects did not show a specific mutation pattern. These results suggest that vaccination failure was likely due to the poor immune response of single individuals and highlights the importance of identifying breakthrough cases and characterizing their clinical and virologic profiles.

Hemagglutinin Gene Variation Rate of H9N2 Avian Influenza Virus by Vaccine Intervention in China.

H9N2 subtype avian influenza virus (AIV) is widespread globally, with China being the main epidemic center. Inactivated virus vaccination was adopted as the main prevention method in China. In this study, 22 hemagglutinin (HA) sequences were obtained from all inactivated vaccine strains of H9N2 subtype AIVs in China since its introduction. A phylogenetic analysis of the vaccine sequences and HA sequences of all published H9N2 subtype AIVs was conducted to investigate the relationship between vaccine use and the virus genetic diversity of the virus. We found that during 2002–2006, when fewer vaccines were used, annual genetic differences between the HA sequences were mainly distributed between 0.025 and 0.075 and were mainly caused by point mutations. From 2009 to 2013, more vaccines were used, and the genetic distance between sequences was about 10 times greater than between 2002 and 2006, especially in 2013. In addition to the accumulation of point mutations, insertion mutations may be the main reason for the large genetic differences between sequences from 2009 to 2013. These findings suggest that the use of inactivated vaccines affected point mutations in the HA sequences and that the contribution of high-frequency replacement vaccine strains to the rate of virus evolution is greater than that of low-frequency replacement vaccine strains. The selection pressure of the vaccine antibody plays a certain role in regulating the variation of HA sequences in H9N2 subtype AIV.

Investigation of cross‐regional spread and evolution of equine influenza H3N8 at US and global scales using Bayesian phylogeography based on balanced subsampling

Equine influenza virus (EIV) is a highly contagious pathogen of equids, and a well-known burden in global equine health. EIV H3N8 variants seasonally emerged and resulted in EIV outbreaks in the United States and worldwide. The present study evaluated the pattern of cross-regional EIV H3N8 spread and evolutionary characteristics at US and global scales using Bayesian phylogeography with balanced subsampling based on regional horse population size. A total of 297 haemagglutinin (HA) sequences of global EIV H3N8 were collected from 1963 to 2019 and subsampled to global subset (n=67), raw US sequences (n=100) and US subset (n=44) datasets. Discrete trait phylogeography analysis was used to estimate the transmission history of EIV using four global and US genome datasets. The North American lineage was the major source of globally dominant EIV variants and spread to other global regions. The US EIV strains generally spread from the southern and midwestern regions to other regions. The EIV H3N8 accumulated approximately three nucleotide substitutions per year in the HA gene under heterogeneous local positive selection. Our findings will guide better decision making of target intervention strategies of EIV H3N8 infection and provide the better scheme of genomic surveillance in the United States and global equine health.

CRISPR/Cas9 unveils the dynamics of the endogenous µ-opioid receptors on neuronal cells under continuous opioidstimulation.

Long‐term opioid use develops tolerance and attenuates analgesic effects. Upon activation, µ‐opioid receptors (MOPs) are internalized and directed to either recycling or degradation pathway. Ligand stimulation also promotes de novo MOP synthesis. These processes collaboratively regulate MOP expression and play critical roles in tolerance development. However, there is limited understanding of how the endogenous MOP expression changes after prolonged opioid administration because previous analyses have focused on individual processes using overexpression systems, which ignored physiological regulation. Another fundamental problem is the unavailability of commercial antibodies to detect the low expression of endogenous MOP in neuronal systems. Here, we established a neuronal cell line to detect endogenous MOP with sufficient sensitivity using CRISPR/Cas9 technology. We incorporated the hemagglutinin sequence into the MOP gene of the SH‐SY5Y cell. The genome‐editing did not significantly impair MOP functions such as MOP internalization or the downstream signaling. The clone was differentiated into a state similar to the primary culture undergoing treatment with all‐trans retinoic acid, followed by brain‐derived neurotrophic factor. Upon continuous stimulation with MOP ligands, endogenous MOP constantly decreased up to 48 h. The expression level was maintained at a certain level following this period, depending on the ligand properties. DAMGO reduced MOP from the cell surface by about 70%, while morphine did so by 40%. Our results indicate that even a few days of opioid administration could significantly reduce the MOP expression level. Our cell line could be a potential tool to investigate the molecular mechanisms underlying the problems caused by long‐term opioid use.

Spatiotemporal Dynamics, Evolutionary History and Zoonotic Potential of Moroccan H9N2 Avian Influenza Viruses from 2016 to 2021.

The H9N2 virus continues to spread in wild birds and poultry worldwide. At the beginning of 2016, the H9N2 Avian influenza virus (AIV) was detected in Morocco for the first time; despite the implementation of vaccination strategies to control the disease, the virus has become endemic in poultry in the country. The present study was carried out to investigate the origins, zoonotic potential, as well as the impact of vaccination on the molecular evolution of Moroccan H9N2 viruses. Twenty-eight (28) H9N2 viruses collected from 2016 to 2021 in Moroccan poultry flocks were isolated and their whole genomes sequenced. Phylogenetic and evolutionary analyses showed that Moroccan H9N2 viruses belong to the G1-like lineage and are closely related to viruses isolated in Africa and the Middle East. A high similarity among all the 2016–2017 hemagglutinin sequences was observed, while the viruses identified in 2018–2019 and 2020–2021 were separated from their 2016–2017 ancestors by long branches. Mutations in the HA protein associated with antigenic drift and increased zoonotic potential were also found. The Bayesian phylogeographic analyses revealed the Middle East as being the region where the Moroccan H9N2 virus may have originated, before spreading to the other African countries. Our study is the first comprehensive analysis of the evolutionary history of the H9N2 viruses in the country, highlighting their zoonotic potential and pointing out the importance of implementing effective monitoring systems.

Sequence Matching between Hemagglutinin and Neuraminidase through Sequence Analysis Using Machine Learning.

To date, many experiments have revealed that the functional balance between hemagglutinin (HA) and neuraminidase (NA) plays a crucial role in viral mobility, production, and transmission. However, whether and how HA and NA maintain balance at the sequence level needs further investigation. Here, we applied principal component analysis and hierarchical clustering analysis on thousands of HA and NA sequences of A/H1N1 and A/H3N2. We discovered significant coevolution between HA and NA at the sequence level, which is closely related to the type of host species and virus epidemic years. Furthermore, we propose a sequence-to-sequence transformer model (S2STM), which mainly consists of an encoder and a decoder that adopts a multi-head attention mechanism for establishing the mapping relationship between HA and NA sequences. The training results reveal that the S2STM can effectively realize the “translation” from HA to NA or vice versa, thereby building a relationship network between them. Our work combines unsupervised and supervised machine learning methods to identify the sequence matching between HA and NA, which will advance our understanding of IAVs’ evolution and also provide a novel idea for sequence analysis methods.

Librator: a platform for the optimized analysis, design, and expression of mutable influenza viral antigens.

Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, limiting their efficiency and utility in high-throughput applications. More streamlined in silico tools allowing researchers to properly analyze and visualize influenza viral protein sequences with accurate nomenclature are necessary to improve antigen design and productivity. To address this need, we developed Librator, a system for analyzing and designing custom protein sequences of influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Within Librator's graphical interface, users can easily interrogate viral sequences and phylogenies, visualize antigen structures and conservation, mutate target residues and design custom antigens. Librator also provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible across related subtypes, thereby promoting reagent savings. Finally, the program facilitates single-cell immune profiling, epitope mapping of monoclonal antibodies and mosaic protein design. Using Librator-based antigen construction, we demonstrate that antigenicity can be readily transferred between HA molecules of H3, but not H1, lineage viruses. Altogether, Librator is a valuable tool for analyzing influenza virus HA and NA proteins and provides an efficient resource for optimizing recombinant influenza antigen synthesis.

Characterization of a 2016-2017 Human Seasonal H3 Influenza A Virus Spillover Now Endemic to U.S. Swine.

ABSTRACTIn 2017, the Iowa State University Veterinary Diagnostic Laboratory detected a reverse-zoonotic transmission of a human seasonal H3 influenza A virus into swine (IAV-S) in Oklahoma. Pairwise comparison between the recently characterized human seasonal H3 IAV-S (H3.2010.2) hemagglutinin (HA) sequences detected in swine and the most similar 2016–2017 human seasonal H3 revealed 99.9% nucleotide identity. To elucidate the origin of H3.2010.2 IAV-S, 45 HA and 27 neuraminidase (NA) sequences from 2017 to 2020 as well as 11 whole-genome sequences (WGS) were genetically characterized. Time to most recent common human ancestor was estimated between August and September 2016. The N2 NA was of human origin in all but one strain from diagnostic submissions with NA sequences, and the internal gene segments from WGS consisted of matrix genes originating from the 2009 pandemic H1N1 and another 5 internal genes of triple reassortant swine origin (TTTTPT). Pigs experimentally infected with H3.2010.2 demonstrated efficient nasal shedding and replication in the lungs, mild pneumonia, and minimal microscopic lung lesions and transmitted the virus to indirect contact swine. Antigenically, H3.2010.2 viruses were closer to a human seasonal vaccine strain, A/Hong Kong/4801/2014, than to the H3.2010.1 human seasonal H3 viruses detected in swine in 2012. This was the second sustained transmission of a human seasonal IAV into swine from the 2010 decade after H3.2010.1. Monitoring the spillover and detection of novel IAV from humans to swine may help vaccine antigen selection and could impact pandemic preparedness.IMPORTANCE H3.2010.2 is a new phylogenetic clade of H3N2 circulating in swine that became established after the spillover of a human seasonal H3N2 from the 2016–2017 influenza season. The novel H3.2010.2 transmitted and adapted to the swine host and demonstrated reassortment with internal genes from strains endemic to pigs, but it maintained human-like HA and NA. It is genetically and antigenically distinct from the H3.2010.1 H3N2 introduced earlier in the 2010 decade. Human seasonal IAV spillovers into swine become established in the population through adaptation and sustained transmission and contribute to the genetic and antigenic diversity of IAV circulating in swine. Continued IAV surveillance is necessary to detect emergence of novel strains in swine and assist with vaccine antigen selection to improve the ability to prevent respiratory disease in swine as well as the risk of zoonotic transmission.

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

BackgroundCanine 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.ResultThe 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.ConclusionsYaH4F 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.Graphical

Phylodynamic study of the conserved RNA structure encompassing the hemagglutinin cleavage site encoding region of H5 and H7 low pathogenic avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIVs) evolve from low pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes. This evolution is characterized by the acquisition of a multi-basic cleavage site (MBCS) motif in the hemagglutinin (HA) that leads to an extended viral tropism and severe disease in poultry. One key unanswered question is whether the risk of transition to HPAIVs is similar for all LPAIVs H5 or H7 strains, or whether specific determinants in the HA sequence of some H5 or H7 LPAIV strains correlate with a higher risk of transition to HPAIVs. Here, we determined if specific features of the conserved RNA stem-loop located at the HA cleavage site-encoding region could be detected along the LPAIV to HPAIV evolutionary pathway. Analysis of the thermodynamic stability of the predicted RNA structures showed no specific patterns common to HA sequences leading to HPAIVs and distinct from those remaining LPAIVs. However, RNA structure clustering analysis revealed that most of the American lineage ancestors leading to H7 emergences via recombination shared the same viral RNA (vRNA) structure topology at the HA1/HA2 boundary region. Our study thus identified predicted secondary RNA structures present in the HA of H7 viruses, which could promote genetic recombination and acquisition of a multibasic cleavage site motif (MBCS).

A Comprehensive Molecular and Epidemiological Characterization of Influenza Viruses Circulating 2016–2020 in North Macedonia

Influenza viruses know no boundaries, representing an example of rapid virus evolution combined with pressure exerted by the host’s immune system. Seasonal influenza causes 4–50 million symptomatic cases in the EU/EEA each year, with a global death toll reaching 650,000 deaths. That being the case, in 2014 North Macedonia introduced the sentinel surveillance in addition to the existing influenza surveillance in order to obtain more precise data on the burden of disease, circulating viruses and to implement timely preventive measures. The aims of this study were to give a comprehensive virological and epidemiological overview of four influenza seasons (2016–2020), assess the frequency and distribution of influenza circulating in North Macedonia and to carry out molecular and phylogenetic analyses of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09, A(H3N2) from ILI and SARI patients. Our results showed that out of 1,632 tested samples, 46.4% were influenza positive, with influenza A(H1N1)pdm09 accounting for the majority of cases (44%), followed by influenza B (32%) and A(H3N2) (17%). By comparing the sentinel surveillance system to the routine surveillance system, we showed that the newly applied system works efficiently and gives great results in the selection of cases. Statistically significant differences (p = < 0.0000001) were observed when comparing the number of reported ILI cases among patients aged 0–4, 5–14, 15–29, and 30–64 years to the reference age group. The phylogenetic analysis of the HA sequences unveiled the resemblance of mutations circulating seasonally worldwide, with a vast majority of circulating viruses belonging to subclade 6B.1A. The PROVEAN analysis showed that the D187A substitution in the receptor binding site (RBS) of the A(H1N1)pdm09 HA has a deleterious effect on the its function. The A(H3N2) viruses fell into the 3C.2a and 3C.3a throughout the analyzed seasons. Molecular characterization revealed that various substitutions in the A(H3N2) viruses gradually replaced the parental variant in subsequent seasons before becoming the dominant variant. With the introduction of sentinel surveillance, accompanied by the advances made in whole-genome sequencing and vaccine therapeutics, public health officials can now modify their approach in disease management and intervene effectively and in a timely manner to prevent major morbidity and mortality from influenza.