H1N1 Virus Research Articles

Evolution and biological characterization of H5N1 influenza viruses bearing the clade 2.3.2.1 hemagglutinin gene

ABSTRACT H5N1 avian influenza viruses bearing the clade 2.3.2.1 hemagglutinin (HA) gene have been widely detected in birds and poultry in several countries. During our routine surveillance, we isolated 28 H5N1 viruses between January 2017 and October 2020. To investigate the genetic relationship of the globally circulating H5N1 viruses and the biological properties of those detected in China, we performed a detailed phylogenic analysis of 274 representative H5N1 strains and analyzed the antigenic properties, receptor-binding preference, and virulence in mice of the H5N1 viruses isolated in China. The phylogenic analysis indicated that the HA genes of the 274 viruses belonged to six subclades, namely clades 2.3.2.1a to 2.3.2.1f; these viruses acquired gene mutations and underwent complicated reassortment to form 58 genotypes, with G43 being the dominant genotype detected in eight Asian and African countries. The 28 H5N1 viruses detected in this study carried the HA of clade 2.3.2.1c (two strains), 2.3.2.1d (three strains), or 2.3.2.1f (23 strains), and formed eight genotypes. These viruses were antigenically well-matched with the H5-Re12 vaccine strain used in China. Animal studies showed that the pathogenicity of the H5N1 viruses ranged from non-lethal to highly lethal in mice. Moreover, the viruses exclusively bound to avian-type receptors and have not acquired the ability to bind to human-type receptors. Our study reveals the overall picture of the evolution of clade 2.3.2.1 H5N1 viruses and provides insights into the control of these viruses.

Evolution of Influenza A(H3N2) Viruses in 2 Consecutive Seasons of Genomic Surveillance, 2021-2023.

The circulation and the genomic evolution of influenza A(H3N2) viruses during the 2021/2022 and 2022/2023 seasons were studied and associated with infection outcomes. Remnant influenza A-positive samples following standard-of-care testing from patients across the Johns Hopkins Health System (JHHS) were used for the study. Samples were randomly selected for whole viral genome sequencing. The sequence-based pEpitope model was used to estimate the predicted vaccine efficacy (pVE) for circulating H3N2 viruses. Clinical data were collected and associated with viral genomic data. A total of 121 683 respiratory specimens were tested for influenza at JHHS between 1 September 2021 and 31 December 2022. Among them, 6071 (4.99%) tested positive for influenza A. Of these, 805 samples were randomly selected for sequencing, with hemagglutinin (HA) segments characterized for 610 samples. Among the characterized samples, 581 were H3N2 (95.2%). Phylogenetic analysis of HA segments revealed the exclusive circulation of H3N2 viruses with HA segments of the 3C.2a1b.2a.2 clade. Analysis of a total of 445 complete H3N2 genomes revealed reassortments; 200 of 227 of the 2022/2023 season genomes (88.1%) were found to have reassorted with clade 3C.2a1b.1a. The pVE was estimated to be -42.53% for the 2021/2022 season and 30.27% for the 2022/2023 season. No differences in clinical presentations or admissions were observed between the 2 seasons. The increased numbers of cases and genomic diversity of influenza A(H3N2) during the 2022/2023 season were not associated with a change in disease severity compared to the previous influenza season.

Spillover of an endemic avian Influenza H6N2 chicken lineage to ostriches and reassortment with clade 2.3.4.4b H5N1 high pathogenicity viruses in chickens

Prior to 2017, chicken production in South Africa had only ever been affected by an endemic strain of H6N2 low pathogenic avian influenza (LPAI), but since 2017, an outbreak of Goose/Guangdong clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) introduced by wild birds, followed by clade 2.3.4.4b H5N1 HPAI (2021-present), affected the country. In the present study, the viruses from seven cases of H6N2 LPAI from commercial poultry between October 2019 and August 2020 were genome-sequenced along with an H5N2 HPAI virus, and phylogenetic analysis was performed. The H5N2 HPAI virus caused localized outbreaks in a small-scale chicken farm and a large commercial layer farm in the KwaZulu-Natal province between late October and early December 2022. The phylogenetic results confirmed the first incidence of the chicken-adapted H6N2 lineage in commercial ostriches in the Western Cape province, with a likely epidemiological origin in chickens from the KwaZulu Natal province. The results also showed that the H5N2 HPAI virus was a novel reassortant of PB2, PB1, PA, NP and NA genome segments derived from a parental H6N2 virus that circulated in region, whereas the HA, M and NS genome segments were derived from sub-genotype SA10 H5N1 HPAI parental virus that had circulated in the local wild bird reservoir since July 2021.

Quasispecies and viral gene expression analysis of the influenza A virus H1N1 strains isolated from human, mallard duck and pig

Quasispecies and viral gene expression analysis of the influenza A virus H1N1 strains isolated from human, mallard duck and pig

Mutual antagonism of mouse-adaptation mutations in HA and PA proteins on H9N2 virus replication

Avian H9N2 viruses have wide host range among the influenza A viruses. However, knowledge of H9N2 mammalian adaptation is limited. To explore the molecular basis of the adaptation to mammals, we performed serial lung passaging of the H9N2 strain A/chicken/Hunan/8.27 YYGK3W3-OC/2018 (3W3) in mice and identified six mutations in the hemagglutinin (HA) and polymerase acidic (PA) proteins. Mutations L226Q, T511I, and A528V of HA were responsible for enhanced pathogenicity and viral replication in mice; notably, HA-L226Q was the key determinant. Mutations T97I, I545V, and S594G of PA contributed to enhanced polymerase activity in mammalian cells and increased viral replication levels in vitro and in vivo. PA-T97I increased viral polymerase activity by accelerating the viral polymerase complex assembly. Our findings revealed that the viral replication was affected by the presence of PA-97I and/or PA-545V in combination with a triple-point HA mutation. Furthermore, the double- and triple-point PA mutations demonstrated antagonistic effect on viral replication when combined with HA-226Q. Notably, any combination of PA mutations, along with double-point HA mutations, resulted in antagonistic effect on viral replication. We also observed antagonism in viral replication between PA-545V and PA-97I, as well as between HA-528V and PA-545V. Our findings demonstrated that several antagonistic mutations in HA and PA proteins affect viral replication, which may contribute to the H9N2 virus adaptation to mice and mammalian cells. These findings can potentially contribute to the monitoring of H9N2 field strains for assessing their potential risk in mammals.

A novel N-heterocycles substituted oseltamivir derivatives as potent inhibitors of influenza virus neuraminidase: discovery, synthesis and biological evaluation

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.

CRP-Tree: a phylogenetic association test for binary traits

Abstract An important problem in evolutionary genomics is to investigate whether a certain trait measured on each sample is associated with the sample phylogenetic tree. The phylogenetic tree represents the shared evolutionary history of the samples and it is usually estimated from molecular sequence data at a locus or from other type of genetic data. We propose a model for trait evolution inspired by the Chinese Restaurant Process that includes a parameter that controls the degree of preferential attachment, that is, the tendency of nodes in the tree to subtend from nodes of the same type. This model with no preferential attachment is equivalent to a structured coalescent model with simultaneous migration and coalescence events and serves as a null model. We derive a test for phylogenetic binary trait association with linear computational complexity and empirically demonstrate that it is more powerful than some other methods. We apply our test to study the phylogenetic association of some traits in swordtail fish, breast cancer, yellow fever virus, and influenza A H1N1 virus. R-package implementation of our methods is available at https://github.com/jyzhang27/CRPTree.

Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system

H7N9 subtype avian influenza virus poses a great challenge for poultry industry. Newcastle disease virus (NDV)-vectored H7N9 avian influenza vaccines (NDVvecH7N9) are effective in disease control because they are protective and allow mass administration. Of note, these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition (HI) but high IgG antibodies in chickens. However, the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear. Herein, immunization with an NDVvecH7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin (HA)-binding IgG antibodies in chickens. Three residues (S150, G151 and S152) in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDVvecH7N9 immune serum. Passively transferred NDVvecH7N9 immune serum conferred complete protection against H7N9 virus infection in chickens. The NDVvecH7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity. These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor, suggesting the engagement of the complement system. Moreover, mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDVvecH7N9 immune serum, indicating the requirement of antibody-antigen binding for complement activity. Therefore, antibodies induced by the NDVvecH7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus. Our findings unveiled a novel role of the complement in protection conferred by the NDVvecH7N9, highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

Reassortant H9N2 canine influenza viruses containing the pandemic H1N1/2009 ribonucleoprotein complex circulating in pigs acquired enhanced virulence in mice

The reassortment between avian H9N2 and Eurasian avian-like (EA) H1N1 viruses may have potentially changed from avian-to-mammals adaptation. This study generated 20 reassortant viruses with the introduction of H1N1/2009 internal genes from EA H1N1 virus into H9N2 virus. 12 of these recovered the replication capability both in the lungs and turbinate samples. 10 of 12 obtained PA gene segments from the ribonucleoprotein (RNP) complexes of the EA H1N1 virus, and 3 exhibited extreme virulence. Specially, the combination of PB2, PA and NP genes could overcome the species-specific restriction in human cells. Analysis of the polymerase activities found that introduction of the PA gene resulted in increased polymerase activity. These findings indicated that RNP complexes from EA H1N1 virus could confer an adaptation advantage and high compatibility to avian H9N2 virus. This raises new concerns for public health due to the possible coexistence of H9N2 and EA H1N1 viruses in dogs.

Mutations in HA and PA affect the transmissibility of H7N9 avian influenza virus in chickens

Low pathogenic (LP) H7N9 avian influenza virus (AIV) emerged in 2013 and had spread widely over several months in China, experienced a noteworthy reduction in isolation rate in poultry and human since 2017. Here, we examined the transmission of H7N9 viruses to better understand viral spread and dissemination mechanisms. Three out of four viruses (2013–2016) could transmit in chickens through direct contact, and airborne transmission was confirmed in the JT157 (2016) virus. However, we did not detect the transmission of the two 2017 viruses, WF69 and AH395, through either direct or airborne exposure. Molecular analysis of genome sequence of two viruses identified eleven mutations located in viral proteins (except for matrix protein), such as PA (K362R and S364N) and HA (D167N, H7 numbering), etc. We explored the genetic determinants that contributed to the difference in transmissibility of the viruses in chickens by generating a series of reassortants in the JT157 background. We found that the replacement of HA gene in JT157 by that of WF69 abrogated the airborne transmission in recipient chickens, whereas the combination of HA and PA replacement led to the loss of airborne and direct contact transmission. Failure with contact transmission of the viruses has been associated with the emergence of the mutations D167N in HA and K362R and S364N in PA. Furthermore, the HA D167N mutation significantly reduced viral attachment to chicken lung and trachea tissues, while mutations K362R and S364N in PA reduced the nuclear transport efficiency and the PA protein expression levels in both cytoplasm and nucleus of CEF cells. The D167N substitution in HA reduced the H7N9 viral acid stability and avian-like receptor binding, while enhanced human-like receptor binding. Further analysis revealed these mutants grew poorly in vitro and in vivo. To conclude, H7N9 AIVs that contain mutations in the HA and PA protein reduced the viral transmissibility in chicken, and may pose a reduced threat for poultry but remain a heightened public health risk.

Drift and shape-new insights into human immunity against influenza virus neuraminidase.

Influenza virus hemagglutinin mediates infection by binding sialic acids, whereas neuraminidase cleaves sialic acids to release progeny virions. Both are targets of protective antibodies, but influenza vaccine strain selection and antigen dose are based on hemagglutinin alone. Virus characterization using first infection ferret sera indicates that escape from hemagglutination inhibiting (HI) antibodies occurs more frequently and is not coordinated with escape from neuraminidase inhibiting (NI) antibodies. A key question addressed by Daulagala et al. (P. Daulagala, B. R. Mann, K. Leung, E. H. Y. Lau, et al., mBio 14:e00084-23, 2023, https://doi.org/10.1128/mbio.00084-23) is how this translates to humans who encounter multiple influenza viruses throughout life. Their cross-sectional study, using sera from a wide age range of participants and H1N1 viruses spanning 1977-2015, indicates that NI antibodies are more broadly cross-reactive than HI antibodies. Both HI and NI titers were highest against strains encountered in childhood indicating that both are shaped by priming exposures. The study further supports the development of NA-optimized vaccines.

Hemagglutinin affects replication, stability and airborne transmission of the H9N2 subtype avian influenza virus

H9N2 subtype avian influenza virus (AIV) can transmit by direct as well as airborne contacts. It has been widespread in poultry and continued to contribute to zoonotic spillover events by providing its six internal genes for the reassortment of novel influenza viruses (eg, H7N9) that infect poultry and humans. Compared to H7N9, H9N2 virus displays an efficient airborne transmissibility in poultry, but the mechanisms of transmission difference have been insufficiently studied. The Hemagglutinin (HA) and viral polymerase acidic protein (PA) have been implicated in the airborne transmission of influenza A viruses. Accordingly, we generated the reassortant viruses of circulating airborne transmissible H9N2 and non-airborne transmissible H7N9 viruses carrying HA and/or PA gene. The introduction of the PA gene from H7N9 into the genome of H9N2 virus resulted in a reduction in airborne transmission among chickens, while the isolated introduction of the HA gene segment completely eliminated airborne transmission among chickens. We further showed that introduction of HA gene of non-transmissible H7N9 did not influence the HA/NA balance of H9N2 virus, but increased the threshold for membrane fusion and decreased the acid stability. Thus, our results indicate that HA protein plays a key role in replication, stability, and airborne transmission of the H9N2 subtype AIV.

Global Prevalence and Hemagglutinin Evolution of H7N9 Avian Influenza Viruses from 2013 to 2022.

H7N9 avian influenza viruses have caused severe harm to the global aquaculture industry and human health. For further understanding of the characteristics of prevalence and hemagglutinin evolution of H7N9 avian influenza viruses, we generated the global epidemic map of H7N9 viruses from 2013 to 2022, constructed a phylogenetic tree, predicted the glycosylation sites and compared the selection pressure of the hemagglutinin. The results showed that although H7N9 avian influenza appeared sporadically in other regions worldwide, China had concentrated outbreaks from 2013 to 2017. The hemagglutinin genes were classified into six distinct lineages: A, B, C, D, E and F. After 2019, H7N9 viruses from the lineages B, E and F persisted, with the lineage B being the dominant. The hemagglutinin of highly pathogenic viruses in the B lineage has an additional predicted glycosylation site, which may account for their persistent pandemic, and is under more positive selection pressure. The most recent ancestor of the H7N9 avian influenza viruses originated in September 1991. The continuous evolution of hemagglutinin has led to an increase in virus pathogenicity in both poultry and humans, and sustained human-to-human transmission. This study provides a theoretical basis for better prediction and control of H7N9 avian influenza.

Towards cost-effective and lightweight surface plasmon resonance biosensing for H5N1 avian influenza virus detection: Integration of novel near-infrared organic photodetectors

H5N1 avian influenza virus (AIV) persists in causing highly fatal human infections, demanding rapid and accurate diagnostic assessment. In this study, we introduce an intensity-based SPR sensor utilizing NIR wavelength excitation in tandem with a specifically engineered NIR-OPD. The active layer of the OPD consists of a PTB7-Th and COTIC-4 F blend, offering an optimized response for a 980 nm excitation wavelength. Key performance metrics of this OPD include a low Jd of 0.185 nA/cm2, a high responsivity of 0.35 A/W, an EQE of 44.74%, and an exceptional detectivity of 4.59 × 1013 Jones at 980 nm wavelength under zero bias. It also exhibits a wide LDR of 113 dB. The integration of such OPDs into our SPR sensor provides advantages in compactness and cost-effectiveness. Employing this sensor, we detected the H5N1 AIV using a custom high-affinity polyclonal antibody against HA envelope of the H5N1 virus, completing analyses of culture medium samples within 12 min. The detection limit of this biosensor for the H5N1 AIV in PBS-diluted culture medium is approximately 4.3 × 104 copies/mL. When compared to a commercial H5-Ag lateral flow test kit, our biosensor showed a sensitivity 37 times higher. Key attributes of our biosensor include 3D printing technology for easy alignment of optical components and a rapid, simplified detection procedure. Collectively, our findings open up the potential of our SPR biosensor as an efficient tool for detecting H5N1 AIV, promising advancements in on-site detection methodologies.

In ovo administration of retinoic acid enhances cell-mediated immune responses against an inactivated H9N2 avian influenza virus vaccine

The H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV that infects avian species and lead to huge economical losses in the poultry industry. The unique immunomodulatory properties of Retinoic acid (RA), an active component of vitamin A, highlights its potential to enhance chicken’s resistance to infectious diseases and perhaps vaccine-induced immunity. Therefore, the present study evaluated the effects of in ovo supplementation of RA on the immunogenicity and protective efficacy of an inactivated avian influenza virus vaccine. On embryonic day 18, eggs were inoculated with either 90 μmol RA/200 μL/egg or diluent into the amniotic sac. On days 7 and 21 post-hatch, birds were vaccinated with 15 μg of β-propiolactone (BPL) inactivated H9N2 virus via the intramuscular route. One group received BPL in combination with an adjuvant, while the other group received saline solution and served as a non-vaccinated control group. Serum samples were collected on days 7, 14, 21, 28, 35, and 42 post-primary vaccination (ppv) for antibody analysis. On day 24 ppv, spleens were collected, and splenocytes were isolated to analyze cytokine expression, interferon gamma (IFN-γ) production, and cell population. On day 28 ppv, birds in all groups were infected with H9N2 virus and oral and cloacal swabs were collected for TCID50 (50 % Tissue Culture Infectious Dose) assay up to day 7 post-infection. The results demonstrated that in ovo administration of RA did not significantly enhance the AIV vaccine-induced antibody response against H9N2 virus compared to the group that received the vaccine alone. However, RA supplementation enhanced the frequency of macrophages (KUL01+), expression of inflammatory cytokines and production of IFN-γ by splenocytes. In addition, RA administration reduced oral shedding of AIV on day 5 post-infection. In conclusion, these findings suggest that RA can be supplemented in ovo to enhance AIV vaccine efficacy against LPAIV.

Inhibition of influenza a virus infection by natural stilbene piceatannol targeting virus hemagglutinin.

Given the magnitude of influenza pandemics as a threat to the global population, it is crucial to have as many prevention and treatment options as possible. Piceatannol (PIC) is a tetrahydroxylated stilbenoid (trans-3,4,3',5'-tetrahydroxystilbene), also known as 3'- hydroxy resveratrol, which has demonstrated many different biological activities such as anti-inflammatory and antiviral activities. In this study, the anti-influenza A virus (IAV) activities and mechanisms of PIC in vitro and in vivo were investigated in order to provide reference for the development of novel plant-derived anti-IAV drugs. The viral plaque assay, RT-PCR and western blot assay were used to evaluate the anti-IAV effects of PIC in vitro. The anti-IAV mechanism of PIC was determined by HA syncytium assay, DARTS assay and Surface Plasmon Resonance assay. The mouse pneumonia model combined with HE staining were used to study the anti-IAV effects of PIC in vivo. PIC shows inhibition on the multiplication of both H1N1 and H3N2 viruses, and blocks the infection of H5N1 pseudovirus with low toxicity. PIC may directly act on the envelope of IAV to induce the rupture and inactivation of IAV particles. PIC can also block membrane fusion via binding to HA2 rather than HA1 and cleavage site of HA0. PIC may interact with the two residues (HA2-T68 and HA2-I75) of HA2 to block the conformational change of HA so as to inhibit membrane fusion. Importantly, oral therapy of PIC also markedly improved survival and reduced viral titers in IAV-infected mice. PIC possesses significant anti-IAV effects both in vitro and in vivo and may block IAV infection mainly through interaction with HA to block membrane fusion. Thus, PIC has the potential to be developed into a new broad-spectrum anti-influenza drug for the prevention and treatment of influenza.

Mutational antigenic landscape of prevailing H9N2 influenza virus hemagglutinin spectrum

H9N2 influenza viruses are globally endemic in birds, and a sharp increase in human infections with H9N2 occurred during 2021 to 2022. In this study, we assess the antigenic and pathogenic impact of 23 hemagglutinin (HA) amino acid mutations. Our study reveals that three specific mutations, labeled R164Q, N166D, and I220T, are responsible for the binding of antibodies with escape mutations. Variants containing R164Q and I220T mutations increase viral replication in avian and mammalian cells. Furthermore, T150A and I220T mutations are found to enhance viral replication in mice, indicating that these mutations may have the potential to adapt mammals. Structure analysis reveals that residues 164 and 220 bearing R164Q and I220T mutations increase interactions with the surrounding residues. Our findings enrich current knowledge about the risk assessment regarding which predominant HA immune-escape mutations of H9N2 viruses may pose the greatest threat to the emergence of pandemics in birds and humans.

Mucosal Administration of Lactobacillus casei Surface-Displayed HA1 Induces Protective Immune Responses against Avian Influenza A Virus in Mice.

Avian influenza is a serious threat to both public health and the poultry industry worldwide. This respiratory virus can be combated by eliciting robust immune responses at the site of infection through mucosal immunization. Recombinant probiotics, specifically lactic acid bacteria, are safe and effective carriers for mucosal vaccines. In this study, we engineered recombinant fusion protein by fusing the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) at the surface of Lactobacillus casei (pgsA-HA1/L. casei). Using subcellular fractionation and flow cytometry we confirmed the surface localization of this fusion protein. Mucosal administration of pgsA-HA1/L. casei in mice resulted in significant levels of HA1-specific serum IgG, mucosal IgA and neutralizing antibodies against the H5N2 virus. Additionally, pgsA-HA1/L. casei-induced systemic and local cell-mediated immune responses specific to HA1, as evidenced by an increased number of IFN-γ and IL-4 secreting cells in the spleens and higher levels of IL-4 in the local lymphocyte supernatants. Finally, mice inoculated with pgsA-HA1/L. casei were protected against a 10LD50 dose of the homologous mouse-adapted H5N2 virus. These results suggest that mucosal immunization with L. casei displaying HA1 on its surface could be a potential strategy for developing a mucosal vaccine against other H5 subtype viruses.

Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from μM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

Portable paper-based molecularly imprinted sensor for visual real-time detection of influenza virus H5N1

The rapid detection of pathogens is important to the control of epidemics. Here, a paper-based molecularly imprinted sensor has been developed for the visual detection of influenza H5N1 virus. Firstly, the paper-based molecularly imprinted was prepared by a sol–gel reaction. Then, H5N1 ∼ Apt was covalently bound to the surface of the ZIF-8-NH2 coated with rhodamine B (RhB) through an amide bond, to produce the fluorescent and colorimetric probe ZIF-8-NH2@RhB ∼ Apt. When H5N1 and ZIF-8-NH2@RhB ∼ Apt are added to the paper-based MIPs, the H5N1 will be combined with MIPs through the synergistic effects of shape and size compatibility and interaction with the functional groups of the monomers, and ZIF-8-NH2@RhB ∼ Apt will bind to H5N1 through its aptamers to form a sandwich structure. The sensor has ultra-high sensitivity and the detection limit is 0.58 fmol/L by using fluorescence spectroscopy. A semi-quantitative signal can also be observed with the naked eye within 10 min. A comparative study showed that direct visual detection was highly reliable, and the sensitivity was better than that achieved with a commercially available kit. Importantly, the paper-based sensor is low in produce cost (Original size: RMB 1.53 Yuan each; Smaller size: RMB 0.08 Yuan each) and is easy to cut, store and carry. This sensor is expected to allow self-service, visual, high-throughput, and real-time virus detection, which is of great significance for epidemic prevention and control.