Hemagglutinin Research Articles

Labeling and isolating cell specific neuronal mitochondria and their functional analysis in mice post stroke.

Dendritic and axonal plasticity, which mediates neurobiological recovery after a stroke, critically depends on the mitochondrial function of neurons. To investigate, in vivo, neuronal mitochondrial function at the stroke recovery stage, we employed Mito-tag mice combined with cerebral cortical infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by two neuronal promoters, synapsin-I (SYN1) and calmodulin-kinase IIa to induce expression of a hemagglutinin (HA)-tagged enhanced green fluorescence protein (EGFP) that localizes to mitochondrial outer membranes of SYN1 positive (SYN+) and CaMKIIa positive (CaMKIIa+) neurons. These mice were then subjected to permanent middle cerebral artery occlusion (MCAO) and sacrificed 14days post stroke. Neuronal mitochondria were then selectively isolated from the fresh brain tissues excised from the ischemic core (IC), ischemic boundary zone (IBZ), as well as from the homologous contralateral hemisphere (CON) by anti-HA magnetic beads for functional analyses. We found that the bead pulled neuronal specific mitochondria were co-precipitated with GFP and enriched with mitochondrial markers, e.g. voltage-dependent anion channel, cytochrome C, and COX IV, but lacked the Golgi protein RCAS1 as well as endoplasmic reticulum markers: Heme‑oxygenase 1 and Calnexin, indicating that specific neuronal mitochondria have been selectively isolated. Western-blot data showed that oxidative phosphorylation (OXPHOS) components in SYN+ and CAMKII+ neuronal mitochondria were significantly decreased in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function indicated by oxygen consumption rate (OCR) (p<0.05, respectively, for both neuron types). These data suggest dysfunction of neuronal mitochondria post stroke is present during the stroke recovery stage. Collectively, for the first time, we demonstrated that using a Mito-tag mouse line combined with AAV9 carrying Cre recombinase approach, neuronal specific mitochondria can be efficiently isolated from the mouse brain to investigate their functional changes post stroke.

Neuraminidase Antibody Response to Homologous and Drifted Influenza A Viruses After Immunization with Seasonal Influenza Vaccines.

Humoral immunity directed against neuraminidase (NA) of the influenza virus may soften the severity of infection caused by new antigenic variants of the influenza viruses. Evaluation of NA-inhibiting (NI) antibodies in combination with antibodies to hemagglutinin (HA) may enhance research on the antibody response to influenza vaccines. The study examined 64 pairs of serum samples from patients vaccinated with seasonal inactivated trivalent influenza vaccines (IIVs) in 2018 according to the formula recommended by the World Health Organization (WHO) for the 2018-2019 flu season. Antibodies against drift influenza viruses A/Guangdong-Maonan/SWL1536/2019(H1N1)pdm09 and A/Brisbane/34/2018(H3N2) were studied before vaccination and 21 days after vaccination. To assess NI antibodies, we used an enzyme-linked lectin assay (ELLA) with pairs of reassortant viruses A/H6N1 and A/H6N2. Anti-HA antibodies were detected using a hemagglutination inhibition (HI) test. The microneutralization (MN) test was performed in the MDCK cell line using viruses A/H6N1 and A/H6N2. Seasonal IIVs induce a significant immune response of NI antibodies against influenza A/H1N1pdm09 and A/H3N2 viruses. A significantly reduced 'herd' immunity to drift influenza A/H1N1pdm09 and A/H3N2 viruses was shown, compared with previously circulating strains. This reduction was most pronounced in strains possessing neuraminidase N2. Seasonal IIVs caused an increase in antibodies against homologous and drifted viruses; however, an increase in antibodies to drifting viruses was observed more often among older patients. The level of NI antibodies for later A/H1N1pdm09 virus in response to IIVs was statistically significantly lower among younger people. After IIV vaccination, the percentage of individuals with HI antibody levels ≥ 1:40 and NI antibody levels ≥ 1:20 was 32.8% for drift A/H1N1pdm09 virus and 17.2% for drift A/H3N2 virus. Antisera containing HI and NI antibodies exhibited neutralizing properties in vitro against viruses with unrelated HA of the H6 subtype. Drift A/H1N1pdm09 and A/H3N2 viruses demonstrated significantly lower reactivity to HI and NI antibodies against early influenza viruses. In response to seasonal IIVs, the level of seroprotection has increased, including against drift influenza A viruses, but protective antibody levels against A/H1N1pdm09 have risen to a greater extent. A reduced immune response to the N1 protein of the A/H1N1pdm09 drift virus was obtained in individuals under 60 years of age. Based on our findings, it is hypothesized that in the cases of a HA mismatch, vaccination against N1-containing influenza viruses may be necessary for individuals under 60, while broader population-level vaccination against N2-containing viruses may be required.

Mitochondrial respiratory capacity in kidney podocytes is high, age-dependent, and sexually dimorphic.

Whether and how podocytes depend on mitochondria across their long post-mitotic lifespan is yet unclear. With limited cell numbers and broad kidney distribution, isolation of podocyte mitochondria typically requires first isolating podocytes themselves. Disassociation of podocytes from their basement membrane, however, recapitulates an injured state that may stress mitochondria. To address this, we crossed floxed hemagglutinin (HA) -mitochondria tagged (MITO-Tag) mice with those expressing Cre in either podocytes (NPHS2) or distal tubule and collecting duct (CDH16), thus allowing for rapid, kidney cell-specific, isolation of mitochondria via immunoprecipitation. Mitochondrial respiration in fresh isolates from young (4-7 mo) and aged (22-26 mo) mice of both sexes demonstrated several previously unreported significant differences between podocyte and tubule mitochondria. First, although podocytes contain fewer mitochondria than do tubule cells, mitochondria isolated from podocytes averaged twice the respiratory capacity of tubule mitochondria when normalized to mitochondrial content by citrate synthase (CS) levels. Second, age-related decline in respiration was detected only in podocyte mitochondria and only in aged male mice. Finally, disassociating podocytes for cell culture initiates functional decline in mitochondria as those from cultured primary podocytes have half the respiratory capacity, but twice the hydrogen peroxide production of podocyte mitochondria isolated directly from fresh kidneys. Thus, podocytes maintain sexually dimorphic mitochondria with greater oxidative phosphorylation capacity than mitochondria-dependent tubules per organelle. Previous studies may not have detected these differences due to reliance on podocyte cell culture conditions, which results in artifactual suppression of mitochondrial function.

Cross-neutralization of Influenza A by SARS-CoV-2 specific neutralizing antibodies and polyclonal plasma: Is pre-exposure to SARS-CoV-2 protective against Influenza A?

According to sparse information from various countries, the seasonal influenza virus circulation has drastically decreased during the COVID-19 pandemic. Here, we show the cross-reactivity of anti-SARS-CoV-2 antibodies against influenza viruses. Plasma samples were collected from 311 SARS-CoV-2 infected individuals. The samples were tested for antibody titers against SARS-CoV-2 by ELISA and seasonal influenza virus strains (influenza A/H1N1, A/H3N2, B/Yamagata, and B/Victoria) using a Hemagglutination Inhibition Assay (HAI). In addition, SARS-CoV-2 antibody-positive but Influenza antibody-negative samples (n = 16) were investigated to determine the SARS-CoV-2 antibody-neutralizing potential against influenza viruses by microneutralization (MN) assay. The SARS-CoV-2 genomes were sequenced using Illumina next-generation sequencing, and an in-silico protein structural analysis was performed to identify epitope and antibody binding similarities between SARS-CoV-2 and influenza viruses. Among 16 samples that didn't contain antibodies against Influenza A strains (H1N1 and H3N2), five showed high (MN titer≥20), and six showed moderate (MN titer≥10) capability to neutralize Influenza A. Subsequent in-silico analysis revealed that most efficient binding (>8 kcal/mol) was found between the antibodies of SARS-CoV-2 delta variant (ΔG) with influenza A/H1N1 HA (Hemagglutinin), A/H3N2 HA, A/H1N1 NA (Neuraminidase), and A/H3N2 NA glycoproteins with −12.4, −9.3, −10.1, and −11.7 kcal/mol, respectively. This investigation revealed that neutralizing antibodies of the delta variant cross-reacted with the Influenza A virus, which might protect against influenza viruses and reduce and shift the seasonal influenza circulation during the COVID-19 pandemic. Our findings warrant further study to explain the probable mechanisms of this cross-reactivity.

Epidemiological and genetic characterization of the influenza A (H1N1) virus in Hangzhou City in 2023.

To explore and describe the epidemiological and genetic variation characteristics of the influenza A (H1N1) virus in Hangzhou City. Respiratory throat swab specimens collected from the fever clinic of the 903rd Hospital of the Chinese People's Liberation Army (PLA) between January and March 2023 were collected. The respiratory pathogen antigens were identified using the colloidal gold method, and those testing positive for influenza A virus antigens were confirmed and subtyped by RT-qPCR. Seventeen H1N1 isolates were selected to amplify hemagglutinin (HA) and neuraminidase (NA) gene sequences via RT-PCR, and sequencing was completed following the identification of the amplified products. The sequenced HA and NA sequences were spliced using DNASTAR software (version 5.0), and a phylogenetic tree was constructed using MEGA software (version 11.0) for genetic characterization. A total of 2,376 respiratory samples were tested, with 680 cases testing positive for influenza A. Of these, 129 positive cases of influenza A were randomly selected for typing, resulting in the isolation of 112 H1N1 subtypes and 17 H3N2 subtypes. The HA genes of 17 strains of influenza A (H1N1) were randomly selected for amino acid homology comparisons with two vaccine strains recommended by the WHO for 2023 (A/Wisconsin/67/2022 (H1N1) and A/Victoria/4897/2022 (H1N1)). The HA gene results showed identities of 98.24 to 98.65% and 98.41 to 98.82%, respectively, and the NA gene results were 98.79 to 99.15% and 98.94 to 99.29%, respectively. Fourteen amino acid sites were altered in the HA gene of the 17 strains, with some strains contributing to the Sa and Ca antigenic determinants, respectively. Seventeen strains had mutations in the NA gene at sites 13, 50, 200, 339, 382, and 469. The sequenced strains, vaccine strains, and some 2023 domestic representative strains independently formed a branch 6B.1A.5a.2a. The continuous evolutionary mutations of the H1N1 virus genes in Hangzhou City suggest the possibility of the virus escaping from the immune response. This study provides an experimental basis for evaluating the protective effect of the vaccine and formulating preventive measures against influenza in Hangzhou City.

Evolutionary analysis of Hemagglutinin and neuraminidase gene variation in H1N1 swine influenza virus from vaccine intervention in China

Influenza poses a significant threat to the global economy and health. Inactivated virus vaccines were introduced in China for prevention in 2018. In this study, three pairs of hemagglutinin (HA) and neuraminidase (NA) gene sequences were obtained from three Swine influenza virus (IAV-S) inactivated vaccine strains that were marketed in China in 2018. Phylogenetic analysis was carried out with HA and NA gene sequences to investigate the relationship between vaccine use and virus genetic drift. The findings showed that the evolutionary rate of HA remained relatively stable from 2012 to 2017, with an average genetic distance of approximately 0.020731195. However, following the introduction of the swine influenza vaccine, there was a notable acceleration in the evolutionary rate of HA, accompanied by a significant increase in the genetic distance. In 2018, the value was 0.111750269, while in 2019 it was 0.176389393. In contrast, the evolution of NA was relatively smooth, with an average genetic distance of approximately 0.030386708. Finally, we demonstrated that commercial vaccines are weak neutralizers of wild strains through immunization experiments in animals. Thus, we have reason to believe that mutations in the virus favor virus evasion of vaccine immunity. Our findings suggest that vaccine use may significantly impact the evolution of the influenza virus by potentially stimulating mutations. The selection pressure of vaccine antibodies played a role in regulating the variation of IAV-S-H1N1.

Unveiling the Role of TMPRSS2 in the Proteolytic Activation of Pandemic and Zoonotic Influenza Viruses and Coronaviruses in Human Airway Cells.

The zoonotic transmission of influenza A viruses (IAVs) and coronaviruses (CoVs) may result in severe disease. Cleavage of the surface glycoproteins hemagglutinin (HA) and spike protein (S), respectively, is essential for viral infectivity. The transmembrane serine protease 2 (TMPRSS2) is crucial for cleaving IAV HAs containing monobasic cleavage sites and severe acute respiratory syndrome (SARS)-CoV-2 S in human airway cells. Here, we analysed and compared the TMPRSS2-dependency of SARS-CoV, Middle East respiratory syndrome (MERS)-CoV, the 1918 pandemic H1N1 IAV and IAV H12, H13 and H17 subtypes in human airway cells. We used the peptide-conjugated morpholino oligomer (PPMO) T-ex5 to knockdown the expression of active TMPRSS2 and determine the impact on virus activation and replication in Calu-3 cells. The activation of H1N1/1918 and H13 relied on TMPRSS2, whereas recombinant IAVs carrying H12 or H17 were not affected by TMPRSS2 knockdown. MERS-CoV replication was strongly suppressed in T-ex5 treated cells, while SARS-CoV was less dependent on TMPRSS2. Our data underline the importance of TMPRSS2 for certain (potentially) pandemic respiratory viruses, including H1N1/1918 and MERS-CoV, in human airways, further suggesting a promising drug target. However, our findings also highlight that IAVs and CoVs differ in TMPRSS2 dependency and that other proteases are involved in virus activation.

Edible bird’s nest: N- and O-glycan analysis and synergistic anti-avian influenza virus activity with neuraminidase inhibitors

Zoonotic avian influenza viruses have continued to infect people on occasion. During treatment, antiviral resistant viruses have occasionally emerged, highlighting the need for a novel strategy for treating human illness. After pancreatin treatment, edible bird's nest (EBN), swiftlet saliva consumed for health purposes, possesses anti-avian viral activity by inhibiting receptor-binding hemagglutinin (HA) activity. Glycan analysis revealed an abundance of α2,3Neu5Ac decoy receptors in pancreatin-treated EBN. Fucosylated tri-α2,3Neu5Ac tri-antennary N-glycans (N-35) and di-α2,3Neu5Ac core 2 O-glycans (O-15) are predominant, accounting for 53.46% and 44.66% of total N- and O-glycan amounts, respectively. Isobologram analysis revealed that the treated EBN had a strong synergistic effect with either oseltamivir carboxylate or zanamivir, a competitive inhibitor of receptor-destroying neuraminidases (NAs), against the avian H5N1 virus. Taken together, EBN has the potential to be developed as a food-derived avian viral trap to prevent and decrease avian virus infection as well as in combination with a viral releasing-NA inhibitor to increase therapeutic potency, reduce toxicity, delay resistance development, and potentially prevent pandemic onset.

Antiviral Effect of Amentoflavone Against Influenza Viruses.

Amentoflavone (AF) is a biflavonoid compound found in many plants. In this study, we first demonstrate that AF has a potent antiviral effect against the influenza virus via the inhibition of viral attachment and virucidal effects. The anti-influenza-viral effect of AF was evaluated using green fluorescent protein-tagged Influenza A virus (IAV) with fluorescent microscopy and flow cytometry analysis. AF decreased the GFP expression by viral infection, dose-dependently. Fifty micromoles of AF suppressed the GFP expression by virus infection of up to 70% of untreated infected control cells. Consistently, immunofluorescence results showed the inhibitory effect of AF on viral protein expression. Time-of-addition and hemagglutination assays revealed that AF inhibits viral binding to cells by interfering with the hemagglutinin (HA) of IAV. Furthermore, AF has a virucidal effect and blocks cytopathic effects caused by the Influenza B virus and H3N2 IAV. Additionally, AF represses the neuraminidase (NA) activity of IAV. In silico analysis confirmed the potential interaction of AF with both HA and NA. Our findings indicate that AF has antiviral effects by modulating HA and NA during the attachment and release stages of influenza viral infection.

Boosting neuraminidase immunity in the presence of hemagglutinin with the next generation of influenza vaccines.

Neuraminidase (NA), the second most abundant surface glycoprotein on the influenza virus, plays a key role in viral replication and propagation. Despite growing evidence showing that NA-specific antibodies correlate with resistance to disease in humans, current licensed vaccines focus almost entirely on the hemagglutinin (HA) antigen. Here, we demonstrate that recombinant NA (rNA) protein is highly immunogenic in both naïve mice and ferrets, as well as in pre-immune ferrets, irrespective of the level of match with preexisting immunity. Ferrets vaccinated with rNA developed mild influenza disease symptoms upon challenge with human H3N2 influenza virus, and anti-NA antibody responses appeared correlated with reduction in disease severity. The addition of rNA to a quadrivalent HA-based vaccine induced robust NA-specific humoral immunity in ferrets, while retaining the ability to induce HA-specific immunity. These results demonstrate that the addition of rNA is a viable option to increase immunogenicity and potentially efficacy versus currently licensed influenza vaccines by means of boosting NA immunity.

Dual receptor-binding, infectivity, and transmissibility of an emerging H2N2 low pathogenicity avian influenza virus.

The 1957 H2N2 influenza pandemic virus [A(H2N2)pdm1957] has disappeared from humans since 1968, while H2N2 avian influenza viruses (AIVs) are still circulating in birds. It is necessary to reveal the recurrence risk and potential cross-species infection of these AIVs from avian to mammals. We find that H2 AIVs circulating in domestic poultry in China have genetic and antigenic differences compared to the A(H2N2)pdm1957. One H2N2 AIV has a dual receptor-binding property similar to that of the A(H2N2)pdm1957. Molecular and structural studies reveal that the N144S, and N144E or R137M substitutions in hemagglutinin (HA) enable H2N2 avian or human viruses to bind or preferentially bind human-type receptor. The H2N2 AIV rapidly adapts to mice (female) and acquires mammalian-adapted mutations that facilitated transmission in guinea pigs and ferrets (female). These findings on the receptor-binding, infectivity, transmission, and mammalian-adaptation characteristics of H2N2 AIVs provide a reference for early-warning and prevention for this subtype.

In vitro Antiviral Activity of Eucheuma cottonii against New Castle Disease Virus

Aims: To determine the antiviral activity of Eucheuma cottonii against the New Castle disease virus. Study Design: In vitro study. Place and Duration of the Study: The research was conducted in the Pharmaceutical Chemistry, Drug Design, and Pharmacognosy department at St. John’s University of Tanzania and Virology Laboratory at Sokoine University of Agriculture between April 2022 and August 2022. Methodology: The present study used an OVO assay and hemagglutination test to investigate the In vitro antiviral activity of E. cottonii collected from Zanzibar, Tanzania, against the Newcastle disease virus (NDV). Ovo assay was assessed using 60 embryonated eggs and a hemagglutination test was performed by collecting the allantoic fluid from treated eggs and serum from hatched chicks to detect antigen titer against NDV using 96 well plate diffusion method. Results: Results of the Ovo assay showed that all the 40 embryonated eggs used as test group were alive and thus confirmed that the extract of E. cottonii (0.32 g/mL) is strong enough to destroy the virus. In which the results from the serial dilution assay (hemagglutination test) confirmed a substantial decrease in the high level of NDV as observed in the allantoic fluid of the infected embryonated eggs to range from 10^-3 to 10^-8 serial dilutions. Conclusion: The OVO assay and hemagglutination results indicate that E. cottonii possesses antiviral activity against NDV. Still further studies like isolation and characterization of bioactive compounds are needed to apply E. cottonii as an alternative source of therapy to fight against NDV.

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.

The highly pathogenic avian influenza H5N8 sublineage 2.3.4.4b virus: A critical analysis of genetic steadiness based on full HA gene sequencing in Egypt

The highly pathogenic avian influenza virus (HPAIV) continues to be a significant threat to the poultry industry despite the implementation of vaccination programs. The main goal of this study was to explore the incidence of avian influenza viruses (AIVs) and the stability of the hemagglutinating (HA) gene sequence. For this drive, oropharyngeal swabs (n=425) were collected from various wild and domesticated bird species from 13 provinces in Egypt from 2019 to 2021. Viral isolation in specific pathogen-free chicken embryos revealed positive HA activity in 9.88% (n=32) of the wild and 36.37% (n=64) of domesticated species. Only eight H5N8 avian influenza viruses were delivered (4/each category) in mixed infection with Newcastle disease virus (NDV). Clinically, domesticated examined birds exhibited clinical findings suggesting avian influenza, but AIVs were delivered from both apparently healthy and sick wild birds. A/chicken-cobb/Egypt/55/2019(H5N8) sequence generated in this study has been submitted to GenBank under the accession number ON724339, and its in vivo pathogenicity was evaluated in four-week-old chickens, which revealed 90% mortality and typical clinical findings of HPAIV. Based on the full HA gene sequence, the strain has a Furin-sensitive cleavage site in the HA protein, which is an HPAIV. It has been clustered in clade 2.3.4.4b within AIVs from 2016 to 2023 and coexistence of genetically stable viruses 2016-2019 with 99.8-100% similarity. The existing strain is fully identical to three strains: A/green-winged teal/Egypt/877/2016(H5N8), A/northern shoveler/Egypt/MB-D-816OP/2016(H5N8), and A/duck/Egypt/N13736E/2017 (H5N8), with the only point of mutation at position 284 (G284E). In our analysis, the majority of H5NX AIV strains detected in Egypt since 2016 possessed 156T. As a result, there is evidence suggesting that H5N8 has been introduced sequentially through the migration of European wild birds along the Black Sea–Mediterranean and East Asia–West Africa flyways. This raises the possibility of future adaptation to mammals due to spillover from avian-origin strains, especially in native aquatic birds such as geese and ducks.

Sequence and Phylogenetic Analysis of Influenza Virus (H1N1pdm2009) Circulating in Riyadh, Saudi Arabia

Influenza A virus (IAV) is the principal cause of seasonal flu and is often reported among pilgrims in Saudi Arabia (SA) due to their mass gatherings. The epidemiological, phylogenetic, and molecular details of A/H1N1pdm2009 in 200 clinical samples collected from hospitalized children in Riyadh during two epidemic seasons (2020/21 and 2021/22) are reported in this study. A total of 21 (10.50%) samples were positive for IAV, as determined using PCR. Fifteen isolates (71.42%) were identified as H1N1pdm2009: eight (53.33%) samples were from males, seven (46.67%) from females. The prevalence of H1N1pdm2009 isolates was significantly (p &lt; 0.05) higher among the age group 15-64 years than the other age groups. A comparison of hemagglutinin (HA) and neuraminidase (NA) amino acid sequences between SA H1N1pdm and certain vaccine strains revealed 19 mutations relative to reference strain A/California/07/2009. Among them, eight (0.47%) were in HA, and eight (0.56%) were in NA sequences that differed from vaccine strains. All isolates of the 2020–2022 seasons exhibited N- and O-glycosylation sites comparable to vaccine strains. Phylogenetically their HA and NA genes are divided into different clades. Most of the studied isolates (five) belonged to clade 5a.1 of HA. These data identify the genetic makeup of circulating influenza virus subtypes.

Hemagglutination-Inhibition Antibodies and Protection against Influenza Elicited by Inactivated and Live Attenuated Vaccines in Children.

Hemagglutinin (HA)-inhibiting antibodies contribute to the immune defense against influenza infection. However, there are insufficient data on the extent of correlation between vaccine-elicited HA antibodies and protection in children against different influenza strains, particularly when comparing live attenuated influenza vaccines (LAIV) versus inactivated influenza vaccines (IIV). We measured postvaccination hemagglutination-inhibition (HAI) titers in 3-15-year-old participants of a cluster-randomized controlled trial of trivalent LAIV(3) versus IIV(3) in Canadian Hutterite colonies. We assessed HAI titers as predictors of symptomatic, reverse transcription polymerase chain reaction (RT-PCR)-confirmed influenza over 3 influenza seasons using Cox proportional hazards regression models with vaccine type as a covariate. For each log2 unit increase in postvaccination HAI against A/H1N1 in 2013-2014, A/H3N2 2014-2015, and B/Yamagata in 2013-2014 (each the predominant circulating strain for the respective influenza season), the reduction in the risk of confirmed influenza was equal to 29.6% (95% confidence interval [CI], 17.1%-39.5%), 34.8% (95% CI, 17.2%-47.9%), and 31.8% (95% CI, 23.8%-38.5%), respectively. No reduction in the risk of influenza was observed with B/Yamagata-specific HAI titers in 2012-2013, which was dominated by a mixture of Yamagata and Victoria strains. Despite the overall lower HAI titers in the LAIV3 group, both H1N1 and H3N2 HAI titers were associated with protection against subtype matched influenza. Both LAIV3- and IIV3-elicited HA antibodies are associated with protection against influenza infection in seasons when the vaccine strains match the circulating influenza strain subtypes, supporting the use of HAI as a correlate of protection for both vaccine types in children.

Molecular Dynamics Investigation of the Influenza Hemagglutinin Conformational Changes in Acidic pH.

The surface protein hemagglutinin (HA) of the influenza virus plays a pivotal role in facilitating viral infection by binding to sialic acid receptors on host cells. Its conformational state is pH-sensitive, impacting its receptor-binding ability and evasion of the host immune response. In this study, we conducted extensive equilibrium microsecond-level all-atom molecular dynamics (MD) simulations of the HA protein to explore the influence of low pH on its conformational dynamics. Specifically, we investigated the impact of protonation on conserved histidine residues (H1062) located in the hinge region of HA2. Our analysis encompassed comparisons between nonprotonated (NP), partially protonated (1P, 2P), and fully protonated (3P) conditions. Our findings reveal substantial pH-dependent conformational alterations in the HA protein, affecting its receptor-binding capability and immune evasion potential. Notably, the nonprotonated form exhibits greater stability compared to protonated states. Conformational shifts in the central helices of HA2 involve outward movement, counterclockwise rotation of protonated helices, and fusion peptide release in protonated systems. Disruption of hydrogen bonds between the fusion peptide and central helices of HA2 drives this release. Moreover, HA1 separation is more likely in the fully protonated system (3P) compared to nonprotonated systems (NP), underscoring the influence of protonation. These insights shed light on influenza virus infection mechanisms and may inform the development of novel antiviral drugs targeting HA protein and pH-responsive drug delivery systems for influenza.

Phytochemical composition and therapeutic potential of Caralluma edulis a cholistani plant

This study explores C. edulis, a plant indigenous to the Cholistan desert, locally known as Pimpa or Seetu, traditionally consumed as a vegetable. Our research aimed to comprehensively analyze its phytochemical constituents, and evaluate its antibacterial, antioxidant, antiviral, anti-inflammatory, antidiabetic, and antipyretic potentials. Utilizing a range of extracts including methanol (MtOH), ethanol (EtOH), ethyl acetate (EA), n-hexane (n-hex), dichloromethane (DCM), and aqueous (Aq), for effective extraction of phytochemicals from C. edulis. Standard biochemical assays and High-Performance Liquid Chromatography-Photodiode Array (HPLC-PDA) were used for analysis of phenolic compounds. Antibacterial effect(s) were confirmed through disc diffusion method and min inhibitory concentrations (MICs). The antioxidant activity was assessed through the Ferric Reducing Antioxidant Power (FRAP) assay and the DPPH radical scavenging method. In vivo antiviral potential was assessed through Hemagglutination (HA) test. Anti-inflammatory, antidiabetic, and antipyretic activities were performed on female albino rats using carrageenan, alloxan monohydrate and yeast-induced methods, respectively. Statistical analysis was done using standard one way ANOVA.Our findings revealed a rich diversity of phenolic compounds and the presence of proteins, alkaloids, and carbohydrates in C. edulis. MtOH and n-hex extracts demonstrated deep antiviral activity against various viral strains. In vivo toxicology studies indicated no significant toxicity at doses up to 5 g/kg. The DCM extract has shown notable anti-inflammatory effects, and EA extract was leading in antipyretic activity. All extracts, except MtOH, exhibited antidiabetic properties.In conclusion, C. edulis emerges not only as a valuable nutritional source but also as a potent alternative medicinal resource, offering wide range of therapeutic benefits.

Conserved sites on the influenza H1 and H3 hemagglutinin recognized by human antibodies.

Monoclonal antibodies (mAbs) targeting the influenza hemagglutinin (HA) have the potential to be used as prophylactics or templates for next-generation vaccines that provide broad protection. Here, we isolated broad, subtype-neutralizing mAbs from human B cells targeting the H1 or H3 HA head as well as a unique mAb targeting the stem. The H1 mAbs target the previously defined lateral patch epitope on H1 HAs and recognize HAs from 1933 to 2021 in addition to a swine H1N1 virus with pandemic potential. Using directed evolution, we improved the neutralization potency of these H1 mAbs towards a contemporary H1 strain. Using deep mutational scanning of four antigenically distinct H1N1 viruses, we identified potential viral escape pathways. For the H3 mAbs we used cryo-EM to define the targeted epitopes: one mAb recognizes the side of the H3 head, accommodating the N133 glycan and a pocket underneath the receptor binding site. The other H3 mAb recognizes an epitope in the HA stem that overlaps with previously characterized mAbs, but with distinct antibody variable genes and mode of recognition. Collectively, these mAbs identify common sites recognized by broad, subtype-specific mAbs that may be elicited by next-generation vaccines.

Antigenicity and genetic properties of an Eurasian avian-like H1N1 swine influenza virus in Jiangsu Province, China

Pigs are vital genetic mixing vessels for human and avian influenza viruses because their tracheal epitheliums possess both sialic acid α-2,6-Gal and α-2,3-Gal receptors. Cross-species transmission of influenza A viruses from swine to humans occurs occasionally. The first case of human infection with the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) genotype G4 was detected in Jiangsu in February 2023, and backtracking epidemiological investigations did not reveal a clear source of the infection. The hemagglutination (HA) and neuraminidase (NA) amino acid variant sites, antiviral drug susceptibility, and antigenic variation of the isolated A/Jiangsu/27271/2023 (JS/27271/23) virus were analyzed, and we evaluated the protective effect of sera collected from occupationally exposed populations in 2024 against the virus. Compared with the vaccine strain, the nucleotide sequence similarities of JS/27271/23 HA and NA were 96.5 % and 95.2 %, respectively. JS/27271/23 was sensitive to polymerase inhibitors (favipiravir and baloxavir), and the antigenicity of its HA protein was 8-fold different from that of the vaccine strain. The percentage of occupationally exposed population with antibody titers of ≥ 40 against A/Hunan/42443/2015 (HN/42443/15) and A/Jiangsu/1/2011 (JS/1/11) were 7.25 % and 2.25 %, respectively, and the geometric mean titers (GMT) were 6.24 and 5.34, respectively. Out of 400 serum samples examined, none had antibody titers of ≥ 40 against JS/27271/23. This suggests that low serum levels of antibodies to EAH1N1 SIVs in occupationally exposed populations may not provide adequate protection because of significant differences in amino acid sites and antigenicity between this virus and the current vaccine strain of EAH1N1 SIVs. There is no evidence of human-to-human transmission of EAH1N1 SIVs. Therefore, surveillance for EAH1N1 SIVs and the development of new vaccine strains are required.