Molecular epidemiology and phylogenetic analysis of influenza viruses A (H3N2) and B/Victoria during the COVID-19 pandemic in Guangdong, China.

BACKGROUND: Non-pharmaceutical measures and travel restrictions have halted the spread of coronavirus disease 2019 (COVID-19) and influenza. Nonetheless, with COVID-19 restrictions lifted, an unanticipated outbreak of the influenza B/Victoria virus in late 2021 and another influenza H3N2 outbreak in mid-2022 occurred in Guangdong, southern China. The mechanism underlying this phenomenon remains unknown. To better prepare for potential influenza outbreaks during COVID-19 pandemic, we studied the molecular epidemiology and phylogenetics of influenza A(H3N2) and B/Victoria that circulated during the COVID-19 pandemic in this region. METHODS: From January 1, 2018 to December 31, 2022, we collected throat swabs from 173,401 patients in Guangdong who had acute respiratory tract infections. Influenza viruses in the samples were tested using reverse transcription-polymerase chain reaction, followed by subtype identification and sequencing of hemagglutinin (HA) and neuraminidase (NA) genes. Phylogenetic and genetic diversity analyses were performed on both genes from 403 samples. A rigorous molecular clock was aligned with the phylogenetic tree to measure the rate of viral evolution and the root-to-tip distance within strains in different years was assessed using regression curve models to determine the correlation. RESULTS: During the early period of COVID-19 control, various influenza viruses were nearly undetectable in respiratory specimens. When control measures were relaxed in January 2020, the influenza infection rate peaked at 4.94% (39/789) in December 2021, with the influenza B/Victoria accounting for 87.18% (34/39) of the total influenza cases. Six months later, the influenza infection rate again increased and peaked at 11.34% (255/2248) in June 2022; influenza A/H3N2 accounted for 94.51% (241/255) of the total influenza cases in autumn 2022. The diverse geographic distribution of HA genes of B/Victoria and A/H3N2 had drastically reduced, and most strains originated from China. The rate of B/Victoria HA evolution (3.11 × 10-3, P < 0.05) was 1.7 times faster than before the COVID-19 outbreak (1.80 × 10-3, P < 0.05). Likewise, the H3N2 HA gene's evolution rate was 7.96 × 10-3 (P < 0.05), which is 2.1 times faster than the strains' pre-COVID-19 evolution rate (3.81 × 10-3, P < 0.05). CONCLUSIONS: Despite the extraordinarily low detection rate of influenza infection, concealed influenza transmission may occur between individuals during strict COVID-19 control. This ultimately leads to the accumulation of viral mutations and accelerated evolution of H3N2 and B/Victoria viruses. Monitoring the evolution of influenza may provide insights and alerts regarding potential epidemics in the future.

MetaFluAD: meta-learning for predicting antigenic distances among influenza viruses.

Influenza viruses rapidly evolve to evade previously acquired human immunity. Maintaining vaccine efficacy necessitates continuous monitoring of antigenic differences among strains. Traditional serological methods for assessing these differences are labor-intensive and time-consuming, highlighting the need for efficient computational approaches. This paper proposes MetaFluAD, a meta-learning-based method designed to predict quantitative antigenic distances among strains. This method models antigenic relationships between strains, represented by their hemagglutinin (HA) sequences, as a weighted attributed network. Employing a graph neural network (GNN)-based encoder combined with a robust meta-learning framework, MetaFluAD learns comprehensive strain representations within a unified space encompassing both antigenic and genetic features. Furthermore, the meta-learning framework enables knowledge transfer across different influenza subtypes, allowing MetaFluAD to achieve remarkable performance with limited data. MetaFluAD demonstrates excellent performance and overall robustness across various influenza subtypes, including A/H3N2, A/H1N1, A/H5N1, B/Victoria, and B/Yamagata. MetaFluAD synthesizes the strengths of GNN-based encoding and meta-learning to offer a promising approach for accurate antigenic distance prediction. Additionally, MetaFluAD can effectively identify dominant antigenic clusters within seasonal influenza viruses, aiding in the development of effective vaccines and efficient monitoring of viral evolution.

Sequence-based detection of emerging antigenically novel influenza A viruses.

The detection of evolutionary transitions in influenza A (H3N2) viruses' antigenicity is a major obstacle to effective vaccine design and development. In this study, we describe Novel Influenza Virus A Detector (NIAViD), an unsupervised machine learning tool, adept at identifying these transitions, using the HA1 sequence and associated physico-chemical properties. NIAViD performed with 88.9% (95% CI, 56.5-98.0%) and 72.7% (95% CI, 43.4-90.3%) sensitivity in training and validation, respectively, outperforming the uncalibrated null model-33.3% (95% CI, 12.1-64.6%) and does not require potentially biased, time-consuming and costly laboratory assays. The pivotal role of the Boman's index, indicative of the virus's cell surface binding potential, is underscored, enhancing the precision of detecting antigenic transitions. NIAViD's efficacy is not only in identifying influenza isolates that belong to novel antigenic clusters, but also in pinpointing potential sites driving significant antigenic changes, without the reliance on explicit modelling of haemagglutinin inhibition titres. We believe this approach holds promise to augment existing surveillance networks, offering timely insights for the development of updated, effective influenza vaccines. Consequently, NIAViD, in conjunction with other resources, could be used to support surveillance efforts and inform the development of updated influenza vaccines.

Adaptation potential of H3N8 canine influenza virus in human respiratory cells.

In 2004, the equine-origin H3N8 canine influenza virus (CIV) first caused an outbreak with lethal cases in racing greyhounds in Florida, USA, and then spread to domestic dogs nationwide. Although transmission of this canine virus to humans has not been reported, it is important to evaluate its zoonotic potential because of the high contact opportunities between companion dogs and humans. To gain insight into the interspecies transmissibility of H3N8 CIV, we tested its adaptability to human respiratory A549 cells through successive passages. We found that CIV acquired high growth properties in these cells mainly through mutations in surface glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). Our reverse genetics approach revealed that HA2-K82E, HA2-R163K, and NA-S18L mutations were responsible for the increased growth of CIV in human cells. Molecular analyses revealed that both HA2 mutations altered the optimum pH for HA membrane fusion activity and that the NA mutation changed the HA-NA functional balance. These findings suggest that H3N8 CIV could evolve into a human pathogen with pandemic potential through a small number of mutations, thereby posing a threat to public health in the future.

Phylogenetic and mutational analysis of H10N3 avian influenza A virus in China: potential threats to human health.

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People's Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient's sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Proposal for a Global Classification and Nomenclature System for A/H9 Influenza Viruses.

Influenza A/H9 viruses circulate worldwide in wild and domestic avian species, continuing to evolve and posing a zoonotic risk. A substantial increase in human infections with A/H9N2 subtype avian influenza viruses (AIVs) and the emergence of novel reassortants carrying A/H9N2-origin internal genes has occurred in recent years. Different names have been used to describe the circulating and emerging A/H9 lineages. To address this issue, an international group of experts from animal and public health laboratories, endorsed by the WOAH/FAO Network of Expertise on Animal Influenza, has created a practical lineage classification and nomenclature system based on the analysis of 10,638 hemagglutinin sequences from A/H9 AIVs sampled worldwide. This system incorporates phylogenetic relationships and epidemiologic characteristics designed to trace emerging and circulating lineages and clades. To aid in lineage and clade assignment, an online tool has been created. This proposed classification enables rapid comprehension of the global spread and evolution of A/H9 AIVs.

The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Occurrence of Circulating Antibodies against the Hemagglutinins of Influenza Viruses in the 2022/2023 Epidemic Season in Poland.

The aim of this study was to determine the level of anti-hemagglutinin antibodies in blood sera collected from patients during the 2022/2023 epidemic season in Poland. A total of 700 sera samples from patients across the country were tested. The samples were divided into seven groups according to the age of the patients, with 100 samples from each age group. The hemagglutination inhibition test (OZHA) was used to determine the level of anti-hemagglutinin antibodies. The test results have confirmed the presence of anti-hemagglutinin antibodies for antigens A/Victoria/2570/2019 (H1N1)pdm09, A/Darwin/9/2021 (H3N2), B/Austria/1359417/2021 (B/Yamagata lineage) and B/ Phuket/3073/2013 (B/Victoria lineage) present in the influenza vaccine recommended by the World Health Organization (WHO) for the 2022/2023 epidemic season. The highest geometric mean antibody titres (GMT) and protection rate values (%) were recorded for hemagglutinin A/H3N2. In Poland, in the 2022/2023 epidemic season, the percentage of the population vaccinated against influenza was 5.7%. Therefore, the test results can be interpreted as the response of the immune system in patients who have been previously infected with an influenza virus.

Self-Assembling Nanoparticle Hemagglutinin Influenza Vaccines Induce High Antibody Response.

As a highly pathogenic avian virus, H5 influenza poses a serious threat to livestock, the poultry industry, and public health security. Hemagglutinin (HA) is both the dominant epitope and the main target of influenza-neutralizing antibodies. Here, we designed a nanoparticle hemagglutinin influenza vaccine to improve the immunogenicity of the influenza vaccine. In this study, HA5 subtype influenza virus was used as the candidate antigen and was combined with the artificially designed double-branch scaffold protein I53_dn5 A and B. A structurally correct and bioactive trimer HA5-I53_dn5B/Y98F was obtained through secretion and purification using an insect baculovirus expression system; I53_dn5A was obtained by purification using a prokaryotic expression system. HA5-I53_dn5B/Y98F and I53_dn5A self-assembled into spherical nanoparticles (HA5-I53_dn5) in vitro with a diameter of about 45 nm. Immunization and serum test results showed that both HA5-I53_dn5B/Y98F and HA5-I53_dn5 could induce HA5-specific antibodies; however, the immunogenicity of HA5-I53_dn5 was better than that of HA5-I53_dn5B/Y98F. Groups treated with HA5-I53_dn5B and HA5-I53_dn5 nanoparticles produced IgG antibody titers that were not statistically different from those of the nanoparticle-containing adjuvant group. This production of trimerized HA5-I53_dn5B and HA5-I53_dn5 nanoparticles using baculovirus expression provides a reference for the development of novel, safe, and efficient influenza vaccines.

Resurgence of influenza with increased genetic diversity of circulating viruses during the 2022-2023 season.

Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.

Multiplex Dual-Target Reverse Transcription PCR for Subtyping Avian Influenza A(H5) Virus.

An increased risk for human infection with avian influenza A(H5N1) viruses is of concern. We developed an internally controlled, dual-target reverse transcription PCR for influenza A(H5) subtyping. This test could be used to detect influenza A(H5) in clinical samples.

Enhancing cross-protection against influenza by heterologous sequential immunization with mRNA LNP and protein nanoparticle vaccines.

Enhancing influenza vaccine cross-protection is imperative to alleviate the significant public health burden of influenza. Heterologous sequential immunization may synergize diverse vaccine formulations and routes to improve vaccine potency and breadth. Here we investigate the effects of immunization strategies on the generation of cross-protective immune responses in female Balb/c mice, utilizing mRNA lipid nanoparticle (LNP) and protein-based PHC nanoparticle vaccines targeting influenza hemagglutinin. Our findings emphasize the crucial role of priming vaccination in shaping Th bias and immunodominance hierarchies. mRNA LNP prime favors Th1-leaning responses, while PHC prime elicits Th2-skewing responses. We demonstrate that cellular and mucosal immune responses are pivotal correlates of cross-protection against influenza. Notably, intranasal PHC immunization outperforms its intramuscular counterpart in inducing mucosal immunity and conferring cross-protection. Sequential mRNA LNP prime and intranasal PHC boost demonstrate optimal cross-protection against antigenically drifted and shifted influenza strains. Our study offers valuable insights into tailoring immunization strategies to optimize influenza vaccine effectiveness.

Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses.

Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01µM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.

Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination.

Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.

Age-dependent heterogeneity in the antigenic effects of mutations to influenza hemagglutinin.

Human influenza virus evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the antigenic effects of viral mutations vary across the human population and how this heterogeneity affects virus evolution. Here, we use deep mutational scanning to map how mutations to the hemagglutinin (HA) proteins of two H3N2 strains, A/Hong Kong/45/2019 and A/Perth/16/2009, affect neutralization by serum from individuals of a variety of ages. The effects of HA mutations on serum neutralization differ across age groups in ways that can be partially rationalized in terms of exposure histories. Mutations that were fixed in influenza variants after 2020 cause greater escape from sera from younger individuals compared with adults. Overall, these results demonstrate that influenza faces distinct antigenic selection regimes from different age groups and suggest approaches to understand how this heterogeneous selection shapes viral evolution.

Exploiting the Affimer platform against influenza A virus.

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli, and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro. Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents. IMPORTANCE: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

Evaluating the epizootic and zoonotic threat of an H7N9 low-pathogenicity avian influenza virus (LPAIV) variant associated with enhanced pathogenicity in turkeys.

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/µL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

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.