Virulent Influenza Virus Research Articles

Two phylogenetic cohorts of the nucleocapsid protein NP and their correlation with the host range of influenza A viruses

Influenza A virus has a wide natural areal among birds, mammals and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin, but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals, including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of avian influenza viruses adaptation to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.

Conserved linear B-cell peptides among the influenza A viral neuraminidases enhance the cross-protective potential of inactivated whole-virion influenza vaccine

Introduction. Influenza is a disease caused by a widespread virus with pandemic potential. Frequently, individuals vaccinated against seasonal influenza virus are still susceptible to the disease, indicating the need to improve the immunogenic potential of existing vaccines. To assess the efficacy of influenza virus vaccines, immune response only to a single viral antigen — hemagglutinin molecule, is taken into consideration. However, according to preclinical and clinical studies, neuraminidase (NA) stimulates cross-protective immunity, which is effective against not only homologous but also drifted variants of influenza A virus. Materials and methods. In the present study, we investigated the ability of previously selected conserved linear B-cell NA epitopes (SGYSGK, SWPDGK, EECSCYPK, VELIRGRK) to enhance the immunogenicity of an inactivated whole-virion influenza vaccine based on the model strain PR8 (iPR8). BALB/c mice were injected with iPR8 in combination with one of the peptides intramuscularly three times at two-week intervals. Blood samples were collected 14 days after the last immunization, after which the mice were challenged with heterosubtypic influenza viruses H1N1pdm09 and H3N2. Results. All immunized mice showed induction of H1N1 (PR8)-specific IgG antibodies two weeks after the third immunization. The group of mice immunized with the iPR8 vaccine preparation in combination with VELIRGRK peptide showed the most pronounced induction of IgG antibodies to the H6N1 reassortant strain, the NA of which corresponds to the iPR8 virus, indicating the ability of the NA peptide to stimulate the production of NA-specific antibodies. However, the antibodies produced after immunization were not capable to inhibit the NA enzymatic activity. Despite this, mice immunized with iPR8 in combination with anti-NA peptides showed a higher survival rate after infection with heterologous virulent influenza viruses: A/California/07/09 (H1N1pdm09) and A/Philippines/2/82 (H3N2) compared to the PBS and iPR8 groups. Conclusion. Thus, the study demonstrated the immune-potentiating effect of individual peptides corresponding to conservative linear epitopes of the NA molecule in combination with a standard inactivated influenza vaccine, which made it possible to improve the protective effect of the vaccine against heterosubtypic influenza viruses.

Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Two Phylogenetic Cohorts of the Nucleocapsid Protein NP and Their Correlation with the Host Range of Influenza A Viruses.

Influenza A virus has a wide natural areal among birds, mammals, and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of adaptation of avian influenza viruses to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.

Ladder distribution of LAIV reassortment between vero adaption attenuated strain and wide type highly virulent influenza virus strain

It described the ladder distribution of Live Attenuated Influenza Vaccine (LAIV) reassortment between Vero cells adaption attenuated strain and wide type high virulent influenza virus strains by the reverse genetics approach. That gene constellation

Anti-influenza A (H1N1) virus effect of gallic acid through inhibition of virulent protein production and association with autophagy.

Influenza remains one of the most serious infectious diseases. Gallic acid is one of the most common and representative phenolic acids found in various plants. This is an interesting subject to explore how gallic acid could inhibit H1N1 influenza virus infection by reducing the production of virulent proteins and interrupting autophagy machinery for influenza virus replication on the host cell. Cellular viability was assessed by XTT assay. The inhibitory effects on the H1N1 influenza virus were assessed by hemagglutination assay, plaque assay, and qRT-PCR. Western blot analysis was used for detecting protein levels of M1, M2, NP, LC3B, and beclin-1. Autophagy activity was demonstrated by acridine orange staining assay. The result demonstrated that there was no cytotoxic effect of gallic acid on A549 cells, and gallic acid could restore the cellular viability of H1N1 influenza virus-infected A549 cells within the experimental concentration treatment. Moreover, gallic acid could effectively restrain viral activity of the H1N1 influenza virus. After the treatment of gallic acid, the production of virulent H1N1 influenza virus proteins, that is, M1, M2, and NP protein were reduced. As for autophagic mechanism, both of the LC3B II conversion and the level ratio of LC3B II to LC3B I were notably decreased. The acridine orange staining assay also revealed decreased accumulation of autophagosomes in H1N1 influenza virus-infected cells. In conclusion, gallic acid suppresses H1N1 influenza viral infectivity through restoration of autophagy pathway and inhibition of virulent M1, M2, and NP protein production.

ViPal: A framework for virulence prediction of influenza viruses with prior viral knowledge using genomic sequences

Influenza viruses pose great threats to public health and cause enormous economic losses every year. Previous work has revealed the viral factors associated with the virulence of influenza viruses in mammals. However, taking prior viral knowledge represented by heterogeneous categorical and discrete information into account to explore virus virulence is scarce in the existing work. How to make full use of the preceding domain knowledge in virulence study is challenging but beneficial. This paper proposes a general framework named ViPal for virulence prediction in mice that incorporates discrete prior viral mutation and reassortment information based on all eight influenza segments. The posterior regularization technique is leveraged to transform prior viral knowledge into constraint features and integrated into the machine learning models. Experimental results on influenza genomic datasets validate that our proposed framework can improve virulence prediction performance over baselines. The comparison between ViPal and other existing methods shows the computational efficiency of our framework with comparable or superior performance. Moreover, the interpretable analysis through SHAP (SHapley Additive exPlanations) identifies the scores of constraint features contributing to the prediction. We hope this framework could provide assistance for the accurate detection of influenza virulence and facilitate flu surveillance.

Utility of HumanIn VitroData in Risk Assessments of Influenza A Virus Using the Ferret Model

As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone.

Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72

Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand (“trans” genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.

Theoretical investigations on the effects of mutations in important residues of NS1B on its RNA-binding using molecular dynamics simulations

NS1B protein plays an important role in countering host antiviral defense and virulence of influenza virus B, considered as the promising target. The first experimental structure of the NS1B protein has recently been determined, was able to bind to double-stranded RNA (dsRNA). However, few studies attempt to investigate the RNA-binding mechanism of the NS1B. In this study, we provide our understanding of the structure-function relationship, dynamics and RNA-binding mechanism of the NS1B protein by performing molecular dynamics simulations combined and MM-GBSA calculations on the NS1B-dsRNA complex. 12 key residues are identified for RNA-binding by forming hydrogen bonds with the. Our results also demonstrate that mutations (R156A, K160A, R208A and K221A) can cause the local structure changes of NS1B CTD and the hydrogen bonds between NS1B CTD and RNA disappearance, which may be the main reasons for the decrease in RNA-binding affinity. These results mentioned will help us understanding the RNA-binding mechanism and could provide some medicinal chemistry insights chances for rational drug design targeting NS1B protein.

Genetic analysis of nucleotide sequences of neuraminidase gene of highly pathogenic avian influenza A/H5N8 virus isolates recovered in the Russian Federation in 2020

Avian influenza is a highly dangerous viral disease that causes huge economic damage to poultry farming. Currently, highly virulent influenza virus with N8 neur- aminidase subtype is quite often detected in populations of domestic and wild birds in various countries of the world. The article provides data on complete nucleotide sequences of the neuraminidase gene of highly pathogenic avian influenza virus isolates recovered in the second half of 2020 from pathological material received from four regions of the Russian Federation. The conducted research showed that the subtype of the isolated virus was N8. According to the phylogenetic analysis, isolates of N8 virus belong to group 8C.4. During the phylogenetic analysis of the neuraminidase, we also took into account data on hemagglutinin classification, according to which H5N8 virus isolates belong to a widespread clade 2.3.4.4. Viruses of the clade were first registered in 2010 in China and they have been circulating up to now. The paper also provides data of a comparative analysis of nucleotide sequences of the studied isolates and the isolates from the international GenBank and GISAID databases, recovered in other countries from 2007 to 2020. During the analysis of the amino acid sequence of the studied isolates, no substitutions were found in the positions that affect resistance to neuraminidase inhibitors. The complete nucleotide sequences of the neuraminidase gene of the avian influenza virus subtype N8 (isolates A/domestic goose/OMSK/1521-1/2020, A/duck/Chelyabinsk/1207-1/2020, A/duck/Saratov/1578-2/2020, A/goose/Tatarstan/1730-2/2020) are published in the international GenBank and GISAID databases. Based on the analysis of the nucleotide sequences of the studied isolates, the article shows gradual evolution of the N8 subtype virus.

Key Amino Acids of M1-41 and M2-27 Determine Growth and Pathogenicity of Chimeric H17 Bat Influenza Virus in Cells and in Mice.

Based on our previous studies, we show that the M gene is critical for the replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing the bat M gene with those from human and swine influenza A viruses. However, the key amino acids of the M1 and/or M2 proteins that are responsible for virus replication and pathogenicity remain unknown. In this study, replacement of the PR8 M gene with the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of the chimeric H17 bat influenza virus. Further studies revealed that M1 was more crucial for viral growth and pathogenicity than M2 and that the amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins identified in this study might be important for influenza virus surveillance and could be used to produce live attenuated vaccines in the future. IMPORTANCE The M1 and M2 proteins influence the morphology, replication, virulence, and transmissibility of influenza viruses. Although a few key residues in the M1 and M2 proteins have been identified, whether other residues of the M1 and M2 proteins are involved in viral replication and pathogenicity remains to be discovered. In the background of the chimeric H17 bat influenza virus, the Eurasian avian-like M gene from the A/California/04/2009 virus significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 in viral growth and pathogenicity in vitro and in vivo and that the key amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine applications in the future. These findings provide important contributions to knowledge of the genetic basis of the virulence of influenza viruses.

Molecular Characterization of Seasonal Influenza A and B from Hospitalized Patients in Thailand in 2018-2019.

Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites (S91R, S181T, T202I) as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission.

The Intersection of Age and Influenza Severity: Utility of Ferrets for Dissecting the Age-Dependent Immune Responses and Relevance to Age-Specific Vaccine Development.

Many factors impact the host response to influenza virus infection and vaccination. Ferrets have been an indispensable reagent for influenza virus research for almost one hundred years. One of the most significant and well-known factors affecting human disease after infection is host age. Another significant factor is the virus, as strain-specific disease severity is well known. Studying age-related impacts on viral infection and vaccination outcomes requires an animal model that reflects both the physiological and immunological changes that occur with human aging, and sensitivity to differentially virulent influenza viruses. The ferret is uniquely susceptible to a plethora of influenza viruses impacting humans and has proven extremely useful in studying the clinical and immunological pictures of influenza virus infection. Moreover, ferrets developmentally have several of the age-related physiological changes that occur in humans throughout infancy, adulthood, old age, and pregnancy. In this review, we discuss ferret susceptibility to influenza viruses, summarize previous influenza studies using ferrets as models of age, and finally, highlight the application of ferret age models in the pursuit of prophylactic and therapeutic agents to address age-related influenza disease severity.

COVID-19 versus H1N1: challenges in radiological diagnosis\u2014comparative study on 130 patients using chest HRCT

BackgroundDuring the current second wave of COVID-19, the radiologists are expected to face great challenges in differentiation between COVID-19 and other virulent influenza viruses, mainly H1N1. Accordingly, this study was performed in order to find any differentiating CT criteria that would help during the expected clinical overlap during the current Influenza season.ResultsThis study was retrospectively conducted during the period from June till November 2020, on acute symptomatic 130 patients with no history of previous pulmonary diseases; 65 patients had positive PCR for COVID-19 including 50 mild patients and 15 critical or severe patients; meanwhile, the other 65 patients had positive PCR for H1N1 including 50 mild patients and 15 critical or severe patients. They included 74 males and 56 females (56.9%:43.1%). Their age ranged 14–90 years (mean age 38.9 ± 20.3 SD). HRCT findings were analyzed by four expert consultant radiologists in consensus. All patients with COVID-19 showed parenchymal or alveolar HRCT findings; only one of them had associated airway involvement. Among the 65 patients with H1N1; 56 patients (86.2%) had parenchymal or alveolar HRCT findings while six patients (9.2%) presented only by HRCT signs of airway involvement and three patients (4.6%) had mixed parenchymal and airway involvement. Regarding HRCT findings of airway involvement (namely tree in bud nodules, air trapping, bronchial wall thickening, traction bronchiectasis, and mucous plugging), all showed significant p value (ranging from 0.008 to 0.04). On the other hand, HRCT findings of parenchymal or alveolar involvement (mainly ground glass opacities) showed no significant relation.ConclusionHRCT can help in differentiation between non-severe COVID-19 and H1N1 based on signs of airway involvement.

Influenza Viruses: Harnessing the Crucial Role of the M2 Ion-Channel and Neuraminidase toward Inhibitor Design.

As a member of the Orthomyxoviridae family of viruses, influenza viruses (IVs) are known causative agents of respiratory infection in vertebrates. They remain a major global threat responsible for the most virulent diseases and global pandemics in humans. The virulence of IVs and the consequential high morbidity and mortality of IV infections are primarily attributed to the high mutation rates in the IVs’ genome coupled with the numerous genomic segments, which give rise to antiviral resistant and vaccine evading strains. Current therapeutic options include vaccines and small molecule inhibitors, which therapeutically target various catalytic processes in IVs. However, the periodic emergence of new IV strains necessitates the continuous development of novel anti-influenza therapeutic options. The crux of this review highlights the recent studies on the biology of influenza viruses, focusing on the structure, function, and mechanism of action of the M2 channel and neuraminidase as therapeutic targets. We further provide an update on the development of new M2 channel and neuraminidase inhibitors as an alternative to existing anti-influenza therapy. We conclude by highlighting therapeutic strategies that could be explored further towards the design of novel anti-influenza inhibitors with the ability to inhibit resistant strains.

N-Linked Glycosylation Plays an Important Role in Budding of Neuraminidase Protein and Virulence of Influenza Viruses.

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knockout significantly inhibited the budding of NA. Further studies showed that the NLG knockout caused attenuation of virus in vitro and in vivo Notably, the NLG at 219 position plays an important role in the budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, the unfolded protein response (UPR) was determined in NLG knockout NA overexpressed cells, which showed that the mutant NA was mainly located in the endoplasmic reticulum (ER), the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knockout NA was stacked in the ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding.IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. In particular, the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10–15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90–94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

Outbreak Severity of Highly Pathogenic Avian Influenza A(H5N8) Viruses Is Inversely Correlated to Polymerase Complex Activity and Interferon Induction.

Highly pathogenic avian influenza A(H5N8) viruses first emerged in China in 2010 and in 2014 spread throughout Asia and to Europe and the United States via migrating birds. Influenza A(H5N8) viruses were first detected in the Netherlands in 2014 and caused five outbreaks in poultry farms but were infrequently detected in wild birds. In 2016, influenza A(H5N8) viruses were reintroduced into the Netherlands, resulting in eight poultry farm outbreaks. This outbreak resulted in numerous dead wild birds with severe pathology. Phylogenetic analysis showed that the polymerase genes of these viruses had undergone extensive reassortment between outbreaks. Here, we investigated the differences in virulence between the 2014-15 and the 2016-17 outbreaks by characterizing the polymerase complex of influenza A(H5N8) viruses from both outbreaks. We found that viruses from the 2014-15 outbreak had significantly higher polymerase complex activity in both human and avian cell lines than did those from the 2016-17 outbreak. No apparent differences in the balance between transcription and replication of the viral genome were observed. Interestingly, the 2014-15 polymerase complexes induced significantly higher levels of interferon beta (IFN-β) than the polymerase complexes of the 2016-17 outbreak viruses, mediated via retinoic acid-inducible gene I (RIG-I). Inoculation of primary duck cells with recombinant influenza A(H5N8) viruses, including viruses with reassorted polymerase complexes, showed that the polymerase complexes from the 2014-15 outbreak induced higher levels of IFN-β despite relatively minor differences in replication capacity. Together, these data suggest that despite the lower levels of polymerase activity, the higher 2016-17 influenza A(H5N8) virus virulence may be attributed to the lower level of activation of the innate immune system.IMPORTANCE Compared to the 2014-15 outbreak, the 2016-17 outbreak of influenza A(H5N8) viruses in the Netherlands and Europe was more virulent; the number of dead or diseased wild birds found and the severity of pathological changes were higher during the 2016-17 outbreak. The polymerase complex plays an important role in influenza virus virulence, and the gene segments of influenza A(H5N8) viruses reassorted extensively between the outbreaks. In this study, the 2014-15 polymerase complexes were found to be more active, which is counterintuitive with the observed higher virulence of the 2016-17 outbreak viruses. Interestingly, the 2014-15 polymerase complexes also induced higher levels of IFN-β. These findings suggest that the higher virulence of influenza A(H5N8) viruses from the 2016-17 outbreak may be related to the lower induction of IFN-β. An attenuated interferon response could lead to increased dissemination, pathology, and mortality, as observed in (wild) birds infected during the 2016-2017 outbreak.

Recombinant Live Attenuated Influenza Vaccine Viruses Carrying Conserved T-cell Epitopes of Human Adenoviruses Induce Functional Cytotoxic T-Cell Responses and Protect Mice against Both Infections.

Human adenoviruses (AdVs) are one of the most common causes of acute respiratory viral infections worldwide. Multiple AdV serotypes with low cross-reactivity circulate in the human population, making the development of an effective vaccine very challenging. In the current study, we designed a cross-reactive AdV vaccine based on the T-cell epitopes conserved among various AdV serotypes, which were inserted into the genome of a licensed cold-adapted live attenuated influenza vaccine (LAIV) backbone. We rescued two recombinant LAIV-AdV vaccines by inserting the selected AdV T-cell epitopes into the open reading frame of full-length NA and truncated the NS1 proteins of the H7N9 LAIV virus. We then tested the bivalent vaccines for their efficacy against influenza and human AdV5 in a mouse model. The vaccine viruses were attenuated in C57BL/6J mice and induced a strong influenza-specific antibody and cell-mediated immunity, fully protecting the mice against virulent influenza virus infection. The CD8 T-cell responses induced by both LAIV-AdV candidates were functional and efficiently killed the target cells loaded either with influenza NP366 or AdV DBP418 peptides. In addition, high levels of recall memory T cells targeted to an immunodominant H2b-restricted CD8 T-cell epitope were detected in the immunized mice after the AdV5 challenge, and the magnitude of these responses correlated with the level of protection against pulmonary pathology caused by the AdV5 infection. Our findings suggest that the developed recombinant vaccines can be used for combined protection against influenza and human adenoviruses and warrant further evaluation on humanized animal models and subsequent human trials.