A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes.

The global spread of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses threatens poultry and public health. The continuous circulation of these viruses has led to their considerable genetic and antigenic evolution, resulting in the formation of eight subclades (2.3.4.4a-h). Here, we examined the antigenic sites that determine the antigenic differences between two H5 vaccine strains, H5-Re8 (clade 2.3.4.4g) and H5-Re11 (clade 2.3.4.4h). Epitope mapping data revealed that all eight identified antigenic sites were located within two classical antigenic regions, with five sites in region A (positions 115, 120, 124, 126, and 140) and three in region B (positions 151, 156, and 185). Through antigenic cartography analysis of mutants with varying numbers of substitutions, we confirmed that a combination of mutations in these eight sites reverses the antigenicity of H5-Re11 to that of H5-Re8, and vice versa. More importantly, our analyses identified H5-Re11_Q115L/R120S/A156T (H5-Re11 + 3) as a promising candidate for a broad-spectrum vaccine, positioned centrally in the antigenic map, and offering potential universal protection against all variants within the clade 2.3.4.4. H5-Re11 + 3 serum has better cross-reactivity than sera generated with other 2.3.4.4 vaccines, and H5-Re11 + 3 vaccine provided 100% protection of chickens against antigenically drifted H5 viruses from various 2.3.4.4 antigenic groups. Our findings suggest that antigenic regions A and B are immunodominant in H5 viruses, and that antigenic cartography-guided vaccine design is a promising strategy for selecting a broad-spectrum vaccine.

The V223I substitution in hemagglutinin reduces the binding affinity to human-type receptors while enhancing the thermal stability of the H3N2 canine influenza virus.

Given the intimate relationship between humans and dogs, the H3N2 canine influenza viruses (CIVs) pose a threat to public health. In our study, we isolated four H3N2 CIVs from 3,758 dog nasal swabs in China between 2018 and 2020, followed by genetic and biological analysis. Phylogenetic analysis revealed 15 genotypes among all available H3N2 CIVs, with genotype 15 prevailing among dogs since around 2017, indicating the establishment of a stable virus lineage in dogs. Molecular characterization identified many mammalian adaptive substitutions, including HA-G146S, HA-N188D, PB2-I292T, PB2-G590S, PB2-S714I, PB1-D154G, and NP-R293K, present across the four isolates. Notably, analysis of HA sequences uncovered a newly emerged adaptive mutation, HA-V223I, which is predominantly found in human and swine H3N2 viruses, suggesting its role in mammalian adaptation. Receptor-binding analysis revealed that the four H3N2 viruses bind both avian and human-type receptors. However, HA-V223I decreases the H3N2 virus's affinity for human-type receptors but enhances its thermal stability. Furthermore, attachment analysis confirmed the H3N2 virus binding to human tracheal tissues, albeit with reduced affinity when the virus carries HA-V223I. Antigenic analysis indicated that the current human H3N2 vaccines do not confer protection against H3N2 CIVs. Collectively, these findings underscore that the potential threat posed by H3N2 CIVs to human health still exists, emphasizing the necessity of close surveillance and monitoring of H3N2 CIVs in dogs.

Does pasteurization inactivate bird flu virus in milk?

Recently, an outbreak of highly pathogenic avian influenza A (H5N1), which carries the clade 2.3.4.4b hemagglutinin (HA) gene and has been prevalent among North American bird populations since the winter of 2021, was reported in dairy cows in the United States. As of 24 May 2024, the virus has affected 63 dairy herds across nine states and has resulted in two human infections. The virus causes unusual symptoms in dairy cows, including an unexpected drop in milk production, and thick colostrum-like milk. Notably, The US Food and Drug Administration reported that around 20% of tested retail milk samples contained H5N1 viruses, with a higher percentage of positive results from regions with infected cattle herds. Data are scant regarding how effectively pasteurization inactivates the H5N1 virus in milk. Therefore, in this study, we evaluated the thermal stability of the H5 clade 2.3.4.4b viruses, along with one human H3N2 virus and other influenza subtype viruses, including H1, H3, H7, H9, and H10 subtype viruses. We also assessed the effectiveness of pasteurization in inactivating these viruses. We found that the avian H3 virus exhibits the highest thermal stability, whereas the H5N1 viruses that belong to clade 2.3.4.4b display moderate thermal stability. Importantly, our data provide direct evidence that the standard pasteurization methods used by dairy companies are effective in inactivating all tested subtypes of influenza viruses in raw milk. Our findings indicate that thermally pasteurized milk products do not pose a safety risk to consumers.

Influenza virus uses mGluR2 as an endocytic receptor to enter cells.

Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy.

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/µL (blue light) and 1.9 × 103 copies/µL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

IGF-1 rs6218 polymorphisms modulate the susceptibility to age-related cataract.

Background: Single nucleotide polymorphisms (SNPs), as the most abundant form of DNA variation in the human genome, contribute to age-related cataracts (ARC) development. Apoptosis of lens epithelial cells (LECs) is closely related to ARC formation. Insulin-like growth factor 1 (IGF1) contributes to cell apoptosis regulation. Moreover, IGF1 was indicated to exhibit a close association with cataract formation. Afterward, an investigation was conducted to examine the correlation between polymorphisms in IGF1 and the susceptibility to ARC. Methods: The present investigation was a case-control study. Venous blood draws were collected from the participants for DNA genotyping. Lens capsule samples were collected to detect mRNA and apoptosis. TaqMan RT-PCR was used to detect IGF1 polymorphism genotypes and qRT PCR was used to detect IGF1 mRNA levels in LECs. LEC apoptosis was evaluated through flow cytometry. The chi-square test was used to compare differences between ARCs and controls of each SNP. Results: We found that the G allele frequency in the IGF1-rs6218 was higher in the ARCs than in the controls. Furthermore, it was observed that the rs6218 GG genotype exhibited a positive correlation to elevated levels of IGF1 mRNA in LECs. The IGF1 mRNA in the LECs and the apoptosis of LECs in nuclear type of ARCs (ARNC) was higher than the controls. Conclusion: The susceptibility to ARC was related to IGF1-rs6218 polymorphism, and this polymorphism is associated with IGF1 expression at the mRNA level. Moreover, apoptosis in LECs of ARNCs was found to be increased.

Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

Correction: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

[This corrects the article DOI: 10.1371/journal.ppat.1006851.].

A responsive hydrogel modulates innate immune cascade fibrosis to promote ocular surface reconstruction after chemical injury.

Following an ocular chemical injury, the release of neutrophil extracellular traps (NETs) triggers an innate immune cascade fibrotic effect involving macrophages (Mø), which limits corneal repair. However, the interplay and mechanisms between NETs and macrophages, as well as the coordination between the innate immunity and corneal repair, remain challenging issues. Using a co-culture system, we report that chemical stimulation exacerbates the accumulation of reactive oxygen species (ROS) within the polymorphonuclear neutrophils, leading to NET formation and the activation of M2 macrophages, ultimately inducing pathological fibrosis of the ocular surface through the IL-10/STAT3/TGF-ß1/Smad2 axis. Inspired by the locally formed acidic microenvironment mediated by innate acute inflammatory stimulation, we further integrate sericin with oxidized chitosan nanoparticles loaded with black phosphorus quantum dots (BPQDs) using Schiff base chemistry to construct a functional pH-responsive hydrogel. Following corneal injury, the hydrogel selectively releases BPQDs in response to the acidic environment, inhibiting the innate immune cascade fibrosis triggered by the PMN-ROS-NETs. Thus, corneal pathological fibrosis is alleviated and reshaping of the ocular surface takes place. These results represent a refinement of the mechanism of inherent immune effector cell interactions, and provide new research ideas for the construction of nano biomaterials that regulate pathological fibrosis.