HA Protein Research Articles

Adaptive amino acid substitutions enhance the virulence of an avian-origin H6N1 influenza virus in mice

The H6N1 subtype avian influenza virus (AIV) is a zoonotic infectious disease pathogen, which poses a threat to human health. In order to study the possible substitution of H6N1 AIV for mammals, an avian-origin H6N1 virus was successively passaged in mice. The results showed that PB2 (L193H and E627K), PA (S709F) and HA (V127I) proteins had multiple amino acid substitutions. The virulence of the mouse-adapted virus was stronger than that of the wild virus, and it was highly pathogenic to mice. Therefore, continued surveillance of these substitutions in poultry H6N1 viruses is required.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 μg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015.

SummaryEquine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub‐lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.

Hydrophobic-interaction chromatography for purification of influenza A and B virus

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved.To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 μg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.

Insights into structural and inhibitory mechanisms of low pH-induced conformational change of influenza HA2 protein: a computational approach.

Though oseltamivir and zanamivir are the active anti-influenza drugs, the emergence of different strains of influenza A virus with mutations creates drug-resistance to these drugs. Therefore, it is essential to find a suitable approach to stop the viral infection. The present study focuses on understanding the conformational changes of the HA2 protein at different pH levels (pH7, pH6, pH5) and on blocking the low pH-induced conformational changes of the HA2 protein with a suitable ligand using molecular docking and molecular dynamics (MD) simulation methods. As the pH value decreases to pH5, the protein undergoes large conformational changes with less stability in the order of pH7 > pH6 > pH5. The fusion peptide (residues 1-20) and the extended loop (residues 58-75) deviate more at pH5. The ligand stachyflin bound between the N- and C-terminal helix regions retains the stability of the HA2 protein at pH5 and blocks the low pH-induced conformational transition. The performance of stachyflin is increased when it directly interacts with residues at the intramonomer binding site rather than the intermonomer binding site. The susceptibility of the HA2 protein of different subtypes to stachyflin is in the order of H1 > H7 > H5 > H2 > H3. Stachflin has a higher binding affinity for H1 (at pH7, pH6, pH5) and H7 subtypes than others. Lys47, Lys58, and Glu103 are the key residues that favor the binding and highly stabilize the HA2 protein at low pH. Graphical abstract Low pH-induced conformational change of influenza HA2 protein.

Subtyping of influenza A H1N1 virus using a label-free electrochemical biosensor based on the DNA aptamer targeting the stem region of HA protein

Rapid subtyping of influenza viruses in clinical laboratories has been increasingly important because three subtypes (seasonal H1N1, H3N2, and 2009 H1N1) of influenza A virus currently disseminated in humans have variable susceptibilities to antiviral drug. Herein, we present DNA aptamers for selective detection of influenza A H1N1 (seasonal and 2009 pandemic H1N1) viruses by targeting recombinant influenza A mini-hemagglutinin (mini-HA) protein (the stable stem region of HA) and whole H1N1 viruses. The dissociation constants (KD) of aptamer candidates V46 and V57 were 19.2 nM and 29.6 nM, respectively, according to electrochemical characterization (differential pulse voltammetry), demonstrating strong binding to mini-HA. In comparison, the KD of the influenza virus antibodies is in the range of 1 μM–10 nM. Aptamer V46 showed higher specificity and binding affinity to the mini-HA protein and H1N1 subtypes, and it was also incorporated into an indium tin oxide-based electrochemical sensor, showing sensitive and specific detection of H1N1 viruses, with a limit of detection (LOD) of 3.7 plaque-forming units per mL. The binding affinity, specificity, and LOD achieved with the electrochemical sensor suggest that it can be used for rapid subtyping of H1N1. We also propose that this aptamer can be used for the neutralization of H1N1 subtypes, suggesting potential therapeutic and diagnostic applications.

Characterization of a strain of quaranfil virus isolated from soft ticks in India. Is quaranfil virus an unrecognized cause of disease in human and animals?”

The soft ticks collected during a field survey in Karnataka state, India, in 1983, yielded a novel virus isolate, which caused mortality in an infant mouse upon inoculation. Attempts at characterizing the virus using the conventional methods were unsuccessful, which prompted us to study it by Next-Generation Sequencing (NGS). This virus isolate was obtained from the viral repository of National Institute of Virology, and an initial virus stock was prepared as a mouse brain homogenate. The virus stock showed cytopathic effects in different cell-lines and was used in NGS. Based on the complete genome sequence, obtained using de novo and reference mapping approach, the virus isolate was identified as a Quaranfil virus (QRFV) belonging to the family Orthomyxoviridae, genus Quaranjavirus. The genome size of the virus is 11,427 nucleotides which consist of 6 segments encoding six proteins. Homology analysis suggested this isolate as similar to QRFV of Afghanistan. In silico analysis showed the HA protein secondary structure to be a class III penetrance similar to Thogotovirus. QRFV was first isolated in 1953 from ticks [Cairo, Egypt] and subsequently reported from other geographical areas. This is the first report describing the presence of QRFV from India. This discovery emphasizes the need for investigating mild febrile illness cases with influenza-like symptoms, particularly in the area of high risk for tick bites.

Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity.

Summary Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz‐t targets the host ubiquitin‐proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz‐t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz‐t, we generated double (K1,2R‐AvrPiz‐t), triple (K1,2,3R‐AvrPiz‐t) and lysine‐free (LF‐AvrPiz‐t) mutants by mutating lysines into arginines in AvrPiz‐t. LF‐AvrPiz‐t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co‐expressed with APIP10 in Nicotiana benthamiana, LF‐AvrPiz‐t was more stable than AvrPiz‐t and was less able to degrade APIP10. The avirulence of LF‐AvrPiz‐t on Piz‐t:HA plants was less than that of AvrPiz‐t, which led to resistance reduction and lower accumulation of the Piz‐t:HA protein after inoculation with the LF‐AvrPiz‐t‐carrying isolate. Chitin‐ and flg22‐induced production of reactive oxygen species (ROS) was higher in LF‐AvrPiz‐t than in AvrPiz‐t transgenic plants. In addition, LF‐AvrPiz‐t transgenic plants were less susceptible than AvrPiz‐t transgenic plants to a virulent isolate. Furthermore, both AvrPiz‐t and LF‐AvrPiz‐t interacted with OsRac1, but the suppression of OsRac1‐mediated ROS generation by LF‐AvrPiz‐t was significantly lower than that by AvrPiz‐t. Together, these results suggest that the lysine residues of AvrPiz‐t are required for its avirulence and virulence functions in rice.

Genetic characteristic of hemagglutinin of avian influenza A (H7N9) virus in Guizhou Province in 2017

The number of H7N9 bird flu cases was high and the situation was grim in guizhou province in 2017. To understand the molecular characteristics of the hemagglutinin gene (HA) and the risk of human infection with avian influenza virus A(H7N9) in Guizhou Province, 2017. Homology, genetic evolution and pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of 14 avian influenza viruses A(H7N9) were analyzed by a series of bioinformation softwares. It was cleared that there was 95.9%-100% similarity among 14 strains in nucleotide of the HA gene, and there were 96.8%-97.8% and 96.8%-97.9% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Phylogenetic analysis showed that 14 HA genes were directly evolved in the Yangtze River Delta evolution branch, but they could be derived from five diffenrent strains. Then 13 of 14 strains cleavage site sequences of HA protein revealed they were low pathogenic avian influenza viruses, while A/Guizhou-Weining/CSY01/2017 was high pathogenic avian influenza virus. Mutation G186V at the receptor binding sites in the HA was found in all 14 strains, and mutation Q226L in 13 strains besides A/Guizhou-Weining/CSY01/2017. All five potential glycosylation motifs in the HA were conservative.

Highly immunogenic influenza virus-like particles containing B-cell-activating factor (BAFF) for multi-subtype vaccine development

Virus-like particle (VLP) technology is an attractive platform for the development of seasonal and pandemic influenza vaccines. Influenza VLPs can be obtained by the overexpression of HA, M1, NA, and/or M2 viral proteins in insect, mammalian, or plant cells. In this study, we reported to obtain highly immunogenic influenza VLPs by molecular incorporation with B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL). Since BAFF and APRIL act as homotrimers to interact with their receptors, we engineered the VLPs by direct fusion of BAFF or APRIL to the transmembrane anchored domain of H5HA gene. Results showed that immunizations with the HA-transmembrane anchored BAFF- or APRIL-VLPs only formulated in alum but not MPL adjuvant elicited significantly higher IgG titers in sera. However, only the BAFF-VLPs formulated in alum adjuvant elicited more broadly neutralizing antibodies against the homologous and two heterologous H5N1 clade/subclade viruses and conferred protective immunity against live virus challenges. As the multi-subtype influenza vaccines containing a variety of HA subtypes can confer broader protective immunity, we also obtained multi-subtype H5H7 BAFF-VLPs and H1H5H7 BAFF-VLPs and demonstrated that these multi-subtype BAFF-VLPs were able to induce the production of neutralizing antibodies against multiple HA subtypes. Our findings provided useful information for the development of highly immunogenic, multi-subtype influenza VLP vaccines.

Multistate design of influenza antibodies improves affinity and breadth against seasonal viruses

Influenza is a yearly threat to global public health. Rapid changes in influenza surface proteins resulting from antigenic drift and shift events make it difficult to readily identify antibodies with broadly neutralizing activity against different influenza subtypes with high frequency, specifically antibodies targeting the receptor binding domain (RBD) on influenza HA protein. We developed an optimized computational design method that is able to optimize an antibody for recognition of large panels of antigens. To demonstrate the utility of this multistate design method, we used it to redesign an antiinfluenza antibody against a large panel of more than 500 seasonal HA antigens of the H1 subtype. As a proof of concept, we tested this method on a variety of known antiinfluenza antibodies and identified those that could be improved computationally. We generated redesigned variants of antibody C05 to the HA RBD and experimentally characterized variants that exhibited improved breadth and affinity against our panel. C05 mutants exhibited improved affinity for three of the subtypes used in design by stabilizing the CDRH3 loop and creating favorable electrostatic interactions with the antigen. These mutants possess increased breadth and affinity of binding while maintaining high-affinity binding to existing targets, surpassing a major limitation up to this point.

Multiple amino acid substitutions involved in the adaption of three avian-origin H7N9 influenza viruses in mice

BackgroundAvian influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. The number of reported H7N9 virus human cases confirmed by laboratory has surpassed that of H5N1 virus. However, the mechanism for how H7N9 influenza virus overcomes host range barrier has not been clearly understood.MethodsTo generate mouse-adapted H7N9 influenza viruses, we passaged three avian-origin H7N9 viruses in mice by lung-to-lung passages independently. Then, the characteristics between the parental and mouse-adapted H7N9 viruses was compared in the following aspects, including virulence in mice, tropism of different tissues, replication in MDCK cells and molecular mutations.ResultsAfter ten passages in mice, MLD50 of the H7N9 viruses reduced >750-3,160,000 folds, and virus titers in MDCK cells increased 10-200 folds at 48 hours post-inoculation. Moreover, the mouse-adapted H7N9 viruses showed more expanded tissue tropism and more serious lung pathological lesions in mice. Further analysis of the amino acids changes revealed 10 amino acid substitutions located in PB2 (E627K), PB1 (W215R and D638G), PA (T97I), HA (H3 numbering: R220G, L226S, G279R and G493R) and NA (P3Q and R134I) proteins. Moreover, PB2 E627K substitution was shared by the three mouse-adapted viruses (two viruses belong to YRD lineage and one virus belongs to PRD lineage), and PA T97A substitution was shared by two mouse-adapted viruses (belong to YRD lineage).ConclusionsOur result indicated that the virulence in mice and virus titer in MDCK cells of H7N9 viruses significantly increased after adapted in mouse model. PB2 E627K and PA T97A substitutions are vital in mouse adaption and should be monitored during epidemiological study of H7N9 virus.

Predicting antigenic variants of H1N1 influenza virus based on epidemics and pandemics using a stacking model.

H1N1 is the earliest emerging subtype of influenza A viruses with available genomic sequences, has caused several pandemics and seasonal epidemics, resulting in millions of deaths and enormous economic losses. Timely determination of new antigenic variants is crucial for the vaccine selection and flu prevention. In this study, we chronologically divided the H1N1 strains into several periods in terms of the epidemics and pandemics. Computational models have been constructed to predict antigenic variants based on epidemic and pandemic periods. By sequence analysis, we demonstrated the diverse mutation patterns of HA1 protein on different periods and that an individual model built upon each period can not represent the variations of H1N1 virus. A stacking model was established for the prediction of antigenic variants, combining all the variation patterns across periods, which would help assess a new influenza strain’s antigenicity. Three different feature extraction methods, i.e. residue-based, regional band-based and epitope region-based, were applied on the stacking model to verify its feasibility and robustness. The results showed the capability of determining antigenic variants prediction with accuracy as high as 0.908 which performed better than any of the single models. The prediction performance using the stacking model indicates clear distinctions of mutation patterns and antigenicity between epidemic and pandemic strains. It would also facilitate rapid determination of antigenic variants and influenza surveillance.

Computational Prediction of the Epitopes of HA1 Protein of Influenza Viruses to its Neutralizing Antibodies

In this work, we have used a new method to predict the epitopes of HA1 protein of influenza virus to several antibodies HC19, CR9114, BH151 and 4F5. While our results reproduced the binding epitopes of H3N2 or H5N1 for the neutralizing antibodies HC19, CR9114, and BH151 as revealed from the available crystal structures, additional epitopes for these antibodies were also suggested. Moreover, the predicted epitopes of H5N1 HA1 for the newly developed antibody 4F5 are located at the receptor binding domain, while previous study identified a region 76-WLLGNP-81 as the epitope. The possibility of antibody recognition of influenza virus via different mechanism by binding to different epitopes of an antigen is also discussed.

Single Immunization with Newcastle Disease Virus-Vectored H7N9 Vaccine Confers a Complete Protection Against Challenge with Highly Pathogenic Avian Influenza H7N9 Virus

In the fifth wave of the H7N9 avian influenza epidemic, highly pathogenic avian influenza (HPAI) A (H7N9) viruses have emerged and pose a great challenge to public health and the poultry industry. In addition, there are apparent genetic and antigenic variations between the classical H7N9 avian influenza virus and the newly-emerged H7N9 virus. Therefore, an antigenic-match vaccine is required for the prevention and control of H7N9 avian influenza in poultry in China. In this study, a recombinant Newcastle disease virus (NDV)-vectored vaccine expressing the HA derived from a prevailing HPAI H7N9 virus (GD15) was generated using reverse genetics. The recombinant virus (rAI4HA) showed virus yield and growth capacity in chicken embryos comparable to the parental virus (rAI4). Expression of the HA protein was detected in chicken embryo fibroblasts inoculated with rAI4HA. A chicken immunization study demonstrated that both rAI4HA and rAI4 induced similar anti-NDV hemagglutination inhibition (HI) antibody titers at weeks 2, 3, and 4 after a single immunization. However, rAI4HA-immunized chickens had a low seroconversion rate (20%) and negative HI titers against H7N9. Additionally, rAI4HA elicited high levels of H7N9-specifc IgY antibody as measured by ELISA. More importantly, the recombinant vaccine provided a complete protection against a lethal challenge with HPAI H7N9 virus and significantly inhibited virus shedding after a single immunization. Our results suggest that the recombinant NDV-vectored H7N9 vaccine expressing the antigenic-match HA can confer a complete protection against HPAI H7N9 challenge after a single immunization.

THE PERFORMANCES OF SOME NEW DENT MAIZE (Zea mays L.) CULTIVARS GROWN AS MAIN CROP IN A MEDITERRANEAN ENVIRONMENT

The experiment was conducted in the experimental fields of at Alasehir district of Manisa province in western Turkey in 2016 and 2017, aiming to determine the adaptability of new maize cultivars for grain production as main crop under Mediterranean ecological conditions. Kayras, Kalends, Kebeos, Keidas, Inove, Performer, Hydro, Pioneer 2088, Pioneer-31-D-24, Pioneer 2105, LG 30600, LG Indaco, LG 30692, BC 678, C-955 and Valbom were tested. Significant differences were detected among the varieties in terms of plant height, first ear height, number of leaves per plant, stem diameter, ear length, ear diameter, number of kernels per ear, 1000 kernel weight, grain yield and crude protein traits. The average value of plant height of maize cultivars varied between 216.03 and 262.45 cm and first ear height 82.70 and 105.48 cm; number of leaves per plant 11.50 and 14.65; stem diameter between 19.0 and 24.1 mm; ear length between 19.33 and 23.06 cm; ear diameter 38.0 to 52.4 mm; number of kernels per ear 314.05 and 563.11; 1000 kernel weight 295.06 and 406.03 g; grain yield 9020.6 kg ha-1 and 12243.0 kg ha-1 and crude protein content between 8.72 and 14.24 %. In conclusion, cv. Pioneer-2105 and cv. Performer were superior than the other cultivars with regard to yield and 1000 kernel weight traits.

Protein Microarray Analysis of the Specificity and Cross-Reactivity of Influenza Virus Hemagglutinin-Specific Antibodies.

Current seasonal influenza virus vaccines engender antibody-mediated protection that is hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA proteins from influenza A virus subtypes H1 to H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1 vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011 influenza season, when H3N2 was prevalent. The response to the H5 vaccine boost was IgG only and confined to H5 variants. The response to natural H3N2 infection consisted of IgG and IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1 proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved epitopes in the stalk domain by cross-reactive antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA antibodies and has many applications in vaccine development.IMPORTANCE Seasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines.

Computational identification of physicochemical signatures for host tropism of influenza A virus.

Avian influenza viruses from migratory birds have managed to cross host species barriers and infected various hosts like human and swine. Epidemics and pandemics might occur when influenza viruses are adapted to humans, causing deaths and enormous economic loss. Receptor-binding specificity of the virus is one of the key factors for the transmission of influenza viruses across species. The determination of host tropism and understanding of molecular properties would help identify the mechanism why zoonotic influenza viruses can cross species barrier and infect humans. In this study, we have constructed computational models for host tropism prediction on human-adapted subtypes of influenza HA proteins using random forest. The feature vectors of the prediction models were generated based on seven physicochemical properties of amino acids from influenza sequences of three major hosts. Feature aggregation and associative rules were further applied to select top 20 features and extract host-associated physicochemical signatures on the combined model of nonspecific subtypes. The prediction model achieved high performance ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mtext>Accuracy</mml:mtext><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>9</mml:mn><mml:mn>4</mml:mn><mml:mn>8</mml:mn></mml:math> , <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mtext>Precision</mml:mtext><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>9</mml:mn><mml:mn>5</mml:mn><mml:mn>4</mml:mn></mml:math> , <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mtext>MCC</mml:mtext><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>9</mml:mn><mml:mn>2</mml:mn><mml:mn>2</mml:mn></mml:math> ). Support and confidence rates were calculated for the host class-association rules. The results indicated that secondary structure and normalized Van der Waals volume were identified as more important physicochemical signatures in determining the host tropism.

Molecular evolutionary and antigenic characteristics of newly isolated H9N2 avian influenza viruses in Guangdong province, China.

Four new H9N2 avian influenza viruses (AIVs) were isolated from domestic birds in Guangdong between December 2015 and April 2016. Nucleotide sequence comparisons indicated that most of the internal genes of these four strains were highly similar to those of human H7N9 viruses. Amino acid substitutions and deletions found in the HA and NA proteins indicated that all four of these new isolates may have an enhanced ability to infect humans and other mammals. A cross-hemagglutinin-inhibition assay, conducted with two vaccine strains that are broadly used in China, suggested that antisera against vaccine candidates could not provide complete inhibition of the new isolates.

A novel recombinant attenuated Newcastle disease virus expressing H9 subtype hemagglutinin protected chickens from challenge by genotype VII virulent Newcastle disease virus and H9N2 avian influenza virus

Newcastle disease virus (NDV) and H9 subtype avian influenza virus (AIV) are two avian pathogens across the globe. Inasmuch as most poultry flocks worldwide are vaccinated with a live low-virulence or attenuated NDV vaccine, we embarked on the development of vaccine prototypes that would have dual specificities and would allow a single immunization against both avian influenza (AI) and Newcastle disease (ND). Therefore, in the present work, a cloned full-length copy of the genome of the lentogenic NDV strain rmNA-1 was selected as a backbone vector to construct three chimeric NDVs that expressed (i) the ORF encoding the HA, (ii) the ectodomain of HA fused with the transmembrane domain and cytoplasmic tail regions derived from the NDV F protein and (iii) the ectodomain of HA fused with a short GS linker and the GCN4 sequences, and designated as rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO), respectively. rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO) stably expressed the modified HA gene for 10 egg passages and the three recombinants were found innocuous to chickens. The insertion of the chimeric HA-F, rather than HA-ECTO or ORF of HA, resulted in a recombinant virus with enhanced incorporation of the HA protein into the viral surface. A single immunization of SPF chickens with the three recombinants induced NDV- and AIV H9-specific antibodies, and protected chickens against a challenge with a lethal dose of velogenic NDV or AIV H9N2. Remarkably, non-shedding of influenza virus and higher levels of H9 subtype HI titers were observed 7 days post challenge (dpc) in rmNA-H9F vaccinated chickens, than other recombinants. Furthermore, a prime-boost vaccination of chickens with rmNA-H9F induced higher levels of NDV- and H9- HI and secretory IgA, as well as reduced viral shedding and virus-induced gross lesions, compared with the commercial vaccine. Therefore, the recombinant rmNA-H9F is a promising bivalent vaccine candidate against NDV and H9 subtype AIV in chickens.