Host proteins interact with viral elements and affect the life cycle of highly pathogenic avian influenza A virus H7N9.

Host-virus interactions can significantly impact the viral life cycle and pathogenesis; however, our understanding of the specific host factors involved in highly pathogenic avian influenza A virus H7N9 (HPAI H7N9) infection is currently restricted. Herein, we designed and synthesized 65 small interfering RNAs targeting host genes potentially associated with various aspects of RNA virus life cycles. Afterward, HPAI H7N9 viruses were isolated and RNA interference was used to screen for host factors likely to be involved in the life cycle of HPAI H7N9. Moreover, the research entailed assessing the associations between host proteins and HPAI H7N9 proteins. Twelve key host proteins were identified: Annexin A (ANXA)2, ANXA5, adaptor related protein complex 2 subunit sigma 1 (AP2S1), adaptor related protein complex 3 subunit sigma 1 (AP3S1), ATP synthase F1 subunit alpha (ATP5A1), COPI coat complex subunit alpha (COP)A, COPG1, heat shock protein family A (Hsp70) member 1A (HSPA)1A, HSPA8, heat shock protein 90 alpha family class A member 1 (HSP90AA1), RAB11B, and RAB18. Co-immunoprecipitation revealed intricate interactions between viral proteins (hemagglutinin, matrix 1 protein, neuraminidase, nucleoprotein, polymerase basic 1, and polymerase basic 2) and these host proteins, presumably playing a crucial role in modulating the life cycle of HPAI H7N9. Notably, ANXA5, AP2S1, AP3S1, ATP5A1, HSP90A1, and RAB18, were identified as novel interactors with HPAI H7N9 proteins rather than other influenza A viruses (IAVs). These findings underscore the significance of host-viral protein interactions in shaping the dynamics of HPAI H7N9 infection, while highlighting subtle variations compared with other IAVs. Deeper understanding of these interactions holds promise to advance disease treatment and prevention strategies.

Enhancing Devulcanizing Degree and Efficiency of Reclaimed Rubber by Using Alcoholic Amines as the Devulcanizing Agent in Low-Temperature Mechano-Chemical Process.

Low-temperature mechanical chemical devulcanization is a process that can produce reclaimed rubber with exceptional mechanical properties. However, the inadequacy and low efficiency of the devulcanization have significantly restricted its application. To address the issues, alcoholic amines, including hydroxyethyl ethylenediamine (AEEA), ethanolamine (ETA), and diethanol amine (DEA), are utilized as devulcanizing agents to promote the devulcanization process. Careful characterizations are conducted to reveal the devulcanizing mechanism and to depict the performances of reclaimed rubbers. Results show that the amine groups in the devulcanizing agents can react with sulfur after the crosslink bonds are broken by mechanical shear force, thus blocking the activity of sulfur and introducing hydroxyl groups into the rubber chains. The incorporation of alcoholic amines can enhance the devulcanizing degree and devulcanizing efficiency, reduce the Mooney viscosity, and improve the mechanical and anti-aging performance. When using DEA as the devulcanizing agent, the sol content of reclaimed rubber increases from 13.1% to 22.4%, the devulcanization ratio increases from 82.1% to 89.0%, the Mooney viscosity decreases from 135.5 to 83.6, the tensile strength improves from 14.7 MPa to 16.3 MPa, the retention rate of tensile strength raises from 55.2% to 82.6% after aging for 72 h, while the devulcanization time is shortened from 21 min to 9.5 min, compared with that without using alcoholic amines. Therefore, alcoholic amines exhibit remarkable advantages in the devulcanization of waste rubber, thus indicating a promising direction for the advancement of research in the area of waste rubber reclamation.

Improving Dispersion of Carbon Nanotubes in Natural Rubber by Using Waterjet-Produced Rubber Powder as a Carrier.

Carbon nanotube (CNT), as reinforcing agents in natural rubber (NR), has gained a large amount of consideration due to their excellent properties. Uniform dispersion of CNT is the key to obtaining high-performance NR nanocomposites. In this contribution, a novel ultrasonic grinding dispersion method of CNT with waterjet-produced rubber powder (WPRP) as a carrier is proposed. Microscopic morphologies show that a Xanthium-like structure with WPRP as the core and CNTs as the spikes is formed, which significantly improves the dispersion of CNT in the NR matrix and simultaneously strengthens the bonding of the WPRP and NR matrix. With the increase in the WPRP loading, the Payne effect of CNT/WPRP/NR composites decreases, indicating the effectiveness of the dispersion method. The vulcanization MH and ML value and crosslinking density increase with the increase in the WPRP loading, whereas the scorch time and cure time exhibit a decreasing trend when the WPRP loading is less than 15 phr. It is found that the CNT/WPRP/NR composites filled with 5 phr WPRP have a 4% increase in 300% modulus, a 3% increase in tensile strength, while a 5% decrease in Akron abrasion loss, compared to CNT/NR composites.

Evaluation of panel of neutralising murine monoclonal antibodies and a humanised bispecific antibody against influenza A(H1N1)pdm09 virus infection in a mouse model.

The influenza A (H1N1) pdm09 virus attracted public attention because of its high prevalence. The annual global morbidity and mortality rates of influenza remain high despite the application of influenza vaccines and antiviral drugs, which indicates the urgent need to identify a more effective strategy for controlling and treating A(H1N1) pdm09 influenza infection. To produce a highly effective therapeutic with broad specificity for A(H1N1) pdm09 influenza viruses, we generated 15 murine monoclonal antibodies (mAbs) via hybridoma technology: 11 mAbs demonstrated 20-100% therapeutic protection in a mouse model of A(H1N1) pdm09 infection at a single dose of 10 mg/kg. A humanised bispecific antibody (Bis-Hu11-1) generated based on the mAbs 3D2 and 3D11, combining the specificities of the two mAbs, was also effective in preventing and treating A(H1N1) pdm09 infection in a mouse model. Bis-Hu11-1 demonstrated hemagglutination inhibition (HI) activity against the escape mutants generated by its parental mAbs that resulted in the obvious reduction in the HI activity of the parental mAbs. In summary, we generated a panel of neutralising mAbs against A(H1N1) pdm09 influenza virus. This study presents a promising method for developing neutralising antibodies that potentially target a series of antigenically diverse influenza viruses.

Recycling of Flexible Polyurethane Foams by Regrinding Scraps into Powder to Replace Polyol for Re-Foaming.

In the context of protecting the ecological environment and carbon neutrality, high-value recycling of flexible polyurethane foam (F-PUF) scraps, generated in the production process, is of great significance to save petroleum raw materials and reduce energy consumption. In the present study, F-PUF scraps were ground into powder by strong shear regrinding using two-roll mill and then reused as a partial replacement of polyol for re-foaming. A series of characterizations were employed to investigate the effect of milling cycles, roller temperatures, and content of the powder on the properties of the powder and F-PUF containing powder. It was revealed that the mechanochemical effect induced breaking of the cross-linking structure and increased activity of the powder. The volume mean diameter (VMD) of powder prepared with 7 milling cycles, at room temperature, is about 97.73 µm. The microstructure and density of the F-PUF containing powder prepared in the above-mentioned manner to replace up to 15 wt.% polyol, is similar to the original F-PUF, with resilience 49.08% and compression set 7.8%, which indicates that the recycling method will play an important role in industrial applications.

Rapid emergence of a PB2 D701N substitution during adaptation of an H9N2 avian influenza virus in mice.

H9N2 avian influenza viruses (AIVs) have been isolated frequently from multiple avian species and, occasionally, from humans. To explore the potential molecular basis of cross-species transmission of H9N2 AIVs, an H9N2 AIV (A/chicken/Zhejiang/221/2016) was serially passaged in mouse lung. The results showed that the mouse-adapted H9N2 virus exhibited higher virulence and replicated more efficiently in mouse lung and liver. Whole-genome sequencing showed an amino acid substitution, D701N, in the PB2 protein, which is likely associated with the increased replicative ability of H9N2 virus in mice. The rapid emergence of adaptive substitutions indicates the necessity of continuous monitoring of H9N2 virus in poultry.

Generation and characterization of monoclonal antibodies against the hemagglutinin of H3N2 influenza A viruses.

Seasonal influenza viruses are highly contagious, leading to 290,000-650,000 mortalities every year globally. Among the influenza viruses, influenza A virus (H3N2) has attracted much attention due to its high frequency of antigenic variations, resulting in poor protection by vaccination. We generated a panel of murine neutralizing monoclonal antibodies (mAbs) against A/Texas/50/2012 (H3N2) and identified the relevant epitopes that potentially influence the antigenicity by selecting mAb-resistant mutants. The epitopes were mainly in antigenic site A (1/9, 11.1%), B (6/9, 66.7%), and C (1/9, 11.1%), which is consistent with recent reports on the immunodominance of antigenic site B. The amino acid substitutions at positions 156, 157, 159, 160, and 189 at antigenic site B resulted in decreased mAb capability for blocking receptor binding. In addition, the neutralizing spectra of three mAbs (1F8, 1G9 and 1H5) were different, suggesting that their epitopes may be different but partially overlapping, and it required further study. Further, the mAb 3F9 selected a new substitution, D53G/N, at antigenic site C and showed in vitro neutralizing activity against A/Victoria/361/2011 (H3N2), A/Texas/50/2012 (H3N2), and A/Hong Kong/2671/2019 (H3N2), suggesting a potential epitope on H3 hemagglutinin for inducing broad neutralizing antibody responses. Continuous research and regular monitoring of novel epitopes are of great importance for improving vaccine strain selection.

Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions.

In this study, a novel multiple-gene reassortant H1N3 subtype avian influenza virus (AIV) (A/chicken/Zhejiang/81213/2017, CK81213) was isolated in Eastern China, whose genes were derived from H1 (H1N3), H7 (H7N3 and H7N9), and H10 (H10N3 and H10N8) AIVs. This AIV belongs to the avian Eurasian-lineage and exhibits low pathogenicity. Serial lung-to-lung passages of CK81213 in mice was performed to study the amino acid substitutions potentially related to the adaptation of H1 AIVs in mammals. And the mouse-adapted H1N3 virus showed greater virulence than wild-type H1N3 AIV in mice and the genomic analysis revealed a total of two amino acid substitutions in the PB2 (E627K) and HA (L67V) proteins. Additionally, the results of the animal study indicate that CK81213 could infect mice without prior adaption and become highly pathogenic to mice after continuous passage. Our findings show that routine surveillance of H1 AIVs is important for the prediction of influenza epidemics.

Generation, characterization, and protective ability of mouse monoclonal antibodies against the HA of A (H1N1) influenza virus.

Influenza virus infections pose a continuous threat to human health. Although vaccines function as a preventive and protective tool, they may not be effective due to antigen drift or an inaccurate prediction of epidemic strains. Monoclonal antibodies (mAbs) have attracted wide attention as a promising therapeutic method for influenza virus infections. In this study, three hemagglutinin (HA)-specific mAbs, named 2A1, 2H4, and 2G2, respectively, were derived from mice immunized with the HA protein from A/Michigan/45/2015(H1N1). The isolated mAbs all displayed hemagglutination inhibition activity and the 2G2 mAb exhibited the strongest neutralization effect. Two amino acid mutations (A198E and G173E), recognized in the process of selection of mAb-resistant mutants, were located in antigenic site Sb and Ca1, respectively. In prophylactic experiments, all three mAbs could achieve 100% protection in mice infected with a lethal dose of A/Michigan/45/2015(H1N1). A dose of 1 mg/kg for 2H4 and 2G2 was sufficient to achieve a full protective effect. Therapeutic experiments showed that all three mAbs could protect mice from death if they received the mAb administration at 6 h postinfection, and 2G2 was still protective after 24 h. Our findings indicate that these three mAbs may have potential prevention and treatment value in an H1N1 epidemic, as well as in the study of antigen epitope recognition.

A multiplex real-time RT-PCR method for detecting H5, H7 and H9 subtype avian influenza viruses in field and clinical samples.

In recent years, H5 and H7 subtypes of highly pathogenic avian influenza viruses (HPAIVs) have been identified in poultry worldwide, resulting in large economic losses to poultry production. Furthermore, H9N2 low pathogenic AIVs are reported to provide internal genes for generating novel reassortant AIVs, leading to potential pandemic risks. To establish an accurate, sensitive and convenient diagnostic method for H5, H7 and H9 subtype AIVs in Eurasian lineage, four groups of specific primers and probes were designed based on the conserved fragments of M, H5, H7 and H9 genes, and a multiplex real-time RT-PCR (RRT-PCR) method was established. High sensitivity was achieved for the multiplex RRT-PCR approach, with a detection limit of 1-10 copies (plasmid DNA) per reaction. The specificity of the method was evaluated using diverse subtypes of AIVs and other avian respiratory viruses isolated in eastern China over the last 9 years. Compared with virus isolation, a higher consistency was achieved when assessing 135 field samples and 126 clinical samples. The results showed that the multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.

Low-Temperature Mechano-Chemical Rubber Reclamation Using Terpinene as a Swelling Agent to Enhance Bond-Breaking Selectivity.

Common swelling agents used in the mechano-chemical rubber devulcanization process usually require high temperatures to achieve satisfactory swelling effects, which results in severe production of pollutants and reduces the selectivity of bond scissions. This work presents an environmentally friendly swelling agent, terpinene, which can swell the rubber crosslink structures at low temperatures. Both a rubber swelling experiment and a rubber reclaiming experiment with a mechano-chemical devulcanization method are conducted to explore the swelling effects of terpinene. After soaking in terpinene at 60 °C for 90 min, the length elongation of the rubber sample reaches 1.55, which is much higher than that in naphthenic oil and is comparable to that in toluene. When adding 3 phr of terpinene for every 100 phr of waste rubber during the reclaiming process, the bond scissions exhibit high selectivity. After revulcanization, the reclaimed rubbers have a tensile strength of 17 MPa and a breaking elongation of 400%. Consequently, the application of terpinene as the swelling agent in the LTMD method can greatly improve the properties of reclaimed rubbers, thereby enhancing the dual value for the economy and environment.

Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies.

BACKGROUND: The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV. METHODS: In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV. RESULTS: The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 µl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 µl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively. CONCLUSIONS: The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.

Molecular characterization and antigenic analysis of reassortant H9N2 subtype avian influenza viruses in Eastern China in 2016.

H9N2 avian influenza viruses (AIVs) can cause respiratory symptoms and decrease the egg production. Additionally, H9N2 AIVs can provide internal genes for reassortment with other subtypes. During the monitoring of live poultry markets in 2016, a total of 32 (32/179, 17.88%) H9N2 AIVs were isolated from poultry in Eastern China, and seven representative strains were selected based on the isolation time, isolation location and sequence homology for further characterization. Phylogenetic analysis of hemagglutinin and neuraminidase showed that these H9N2 AIVs clustered into the Y280 sublineage. And the phylogenetic trees of six internal genes showed that the source of these gene fragments was more abundant, suggesting that extensive reassortment has occurred in these H9N2 viruses. Molecular analysis showed that multiple specific amino acid mutations occurred that increased H9N2 AIVs' infectivity, transmissibility, and affinity to mammals, including Q226L and Q227M in hemagglutinin, E627K in polymerase basic protein 2 (PB2), L13P in polymerase basic protein 1 (PB1), and A70V and S409N in polymerase acidic protein (PA). Pathogenicity tests in mice showed these H9N2 AIVs could replicate in lungs and exhibited slight to moderate virulence. The continuous circulation of these H9N2 viruses suggests the necessity for persistent surveillance of the H9N2 AIVs in poultry.

Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study.

There is a worldwide pandemic of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; yet our understanding remains limited on the characteristic of antibodies, especially for dynamic long-term tracking. Sequential serum samples were collected up to 416 days post onset of symptoms (POS) from 102 patients who were hospitalized with coronavirus disease 2019 (COVID-19). Immunoglobulin (Ig)G, IgM, and IgA levels targeting SARS-CoV-2 spike 1 receptor-binding domain (S1-RBD), spike 2 extracellular domain (S2-ECD), and nucleocapsid protein (N) were quantified as well as neutralizing activity. We were pleasantly surprised to find that the antibody remained detective and effective for more than a year POS. We also found the varied reactions of different antibodies as time passed: N-IgA rose most rapidly in the early stage of infection, while S2-IgG was present at a high level in the long time of observation. This study described the long traceable antibody response of the COVID-19 and offered hints about targets to screen for postinfectious immunity and for vaccination development of SARS-CoV-2.

Genetic analysis and biological characteristics of novel clade 2.3.4.4 reassortment H5N6 avian influenza viruses from poultry in eastern China in 2016.

OBJECTIVES: The continuous evolution of highly pathogenic H5N6 avian influenza viruses (AIVs) causes outbreaks in wildfowl and poultry, and occasional human infections. The aim of this study was to better understand the genetic relationships between these H5N6 AIVs from eastern China and other AIVs. METHODS: In 2016, 1623 cloacal swabs were sampled from poultry in 18 LPMs in eastern China, and subsequently characterized systematically using gene sequencing, phylogenetic studies, and antigenic analysis. In addition, their pathogenicity in mammals was studied in BALB/c mice, which were inoculated with viruses, with survival rate and body weight recorded daily for 14 days. RESULTS: In total, 56 H5N6 AIVs were isolated in eastern China and five representative isolates were selected for further study. In our study, the H5N6 AIVs clustered into clade 2.3.4.4, Group C, and their six internal segments were derived from H6N6 and H9N2 viruses, or both, suggesting extensive reassortant among H5N6 AIVs with other subtypes. These H5N6 viruses could replicate in the lungs without prior adaptation, and exhibited slight-to-moderate virulence in mice. CONCLUSIONS: The continuous circulation of these novel H5N6 viruses suggests the importance of persistent surveillance of H5N6 AIVs in poultry.

Development of an antigen-ELISA and a colloidal gold-based immunochromatographic strip based on monoclonal antibodies for detection of avian influenza A(H5) viruses.

Avian influenza A(H5) viruses (avian IAVs) pose a major threat to the economy and public health. We developed an antigen-ELISA (ag-ELISA) and a colloidal gold-based immunochromatographic strip for the rapid detection of avian A(H5) viruses. Both detection methods displayed no cross-reactivity with other viruses (e.g., other avian IAVs, infectious bursal disease virus, Newcastle disease virus, infectious bronchitis virus, avian paramyxovirus). The ag-ELISA was sensitive down to 0.5 hemagglutinin (HA) units/100 µL of avian A(H5) viruses and 7.5 ng/mL of purified H5 HA proteins. The immunochromatographic strip was sensitive down to 1 HA unit/100 µL of avian A(H5) viruses. Both detection methods exhibited good reproducibility with CVs < 10%. For 200 random poultry samples, the sensitivity and specificity of the ag-ELISA were 92.6% and 98.8%, respectively, and for test strips were 88.9% and 98.3%, respectively. Both detection methods displayed high specificity, sensitivity, and stability, making them suitable for rapid detection and field investigation of avian A(H5) viruses.

Methylene blue photochemical treatment as a reliable SARS-CoV-2 plasma virus inactivation method for blood safety and convalescent plasma therapy for COVID-19.

BACKGROUND: In 2020, a new coronavirus, SARS-CoV-2, quickly spread worldwide within a few months. Although coronaviruses typically infect the upper or lower respiratory tract, the virus RNA can be detected in plasma. The risk of transmitting coronavirus via transfusion of blood products remains. As more asymptomatic infections are identified in COVID-19 cases, blood safety has become particularly important. Methylene blue (MB) photochemical technology has been proven to inactivate lipid-enveloped viruses with high efficiency and safety. The present study aimed to investigate the SARS-CoV-2 inactivation effects of MB in plasma. METHODS: The SARS-CoV-2 virus strain was isolated from Zhejiang University. The live virus was harvested from cultured VERO-E6 cells, and mixed with MB in plasma. The MB final concentrations were 0, 1, 2, and 4 µM. The "BX-1 AIDS treatment instrument" was used at room temperature, the illumination adjusted to 55,000 ± 0.5 million Lux, and the plasma was irradiated for 0, 2, 5, 10, 20, and 40 mins using light at a single wavelength of 630 nm. Virus load changes were measured using quantitative reverse transcription- PCR. RESULTS: BX-1 could effectively eliminate SARS-CoV-2 within 2 mins in plasma, and the virus titer declined to 4.5 log10 TCID50 (median tissue culture infectious dose)/mL. CONCLUSION: BX-1 is based on MB photochemical technology, which was designed to inactivate HIV-1 virus in plasma. It was proven to be safe and reliable in clinical trials of HIV treatment. In this study, we showed that BX-1 could also be applied to inactivate SARS-CoV-2. During the current outbreak, this technique it has great potential for ensuring the safety of blood transfusions, for plasma transfusion therapy in recovering patients, and for preparing inactivated vaccines.

Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses.

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.