Hemagglutinin glycosylation pattern-specific effects: implications for the fitness of H9.4.2.5-branched H9N2 avian influenza viruses.

Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

Potential of Lactobacillus plantarum A56 in relieving food allergy through immunoregulation, antioxidation, and reshaping intestinal microbiota.

Food allergy is an abnormal immune reaction triggered by food protein antigens. Relevant studies have suggested that probiotic supplementation was with the potential to alleviate food allergy. This study aimed to explore the effects of Lactobacillus plantarum A56 on the alleviation of ovalbumin (OVA)-induced food allergy via immunomodulatory function, antioxidation, and modification of intestinal microbiota. Balb/c mice were sensitized with OVA (20 µg/mouse) by intraperitoneal injection for 3 weeks and accompanied by oral administration of L. plantarum A56 (109 CFU/mL), subsequently with orally challenged twice by OVA at 50 mg/mL for 1 week. The results showed that oral supplementation of L. plantarum A56 could effectively relieve allergic symptoms of mice, and decreased OVA-specific IgE and IgG1 concentrations. It also declined interleukin (IL)-4 level, raised interferon-γ (IFN-γ) in serum, and splenocyte supernatant, and the qPCR results were consistent with above results. Moreover, L. plantarum A56 treatment also fortified superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels, and reduced malondialdehyde (MDA) level in serum. The increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and forkhead box O1 (Foxo1) expression indicated that L. plantarum A56 exerted antioxidation through Nrf2-Foxo1 pathway. In addition, L. plantarum A56 treatment elevated Bacteroidetes richness, ASV/OTU number, species diversity, etc. Notably, Spearman correlation analysis indicated that Bacteroidetes displayed obviously negative correlation with IgE and IgG1, but Actinobacteria and Acidobacteria exhibited significantly positive correlation with IgG1 and IgE. Collectively, these results suggested that L. plantarum A56 could alleviate OVA-induced food allergy by regulating Th1/Th2 imbalance, antioxidation, and modulating intestinal microbiota.

Development and Immunoprotection of Bacterium-like Particle Vaccine against Infectious Bronchitis in Chickens.

Infectious bronchitis (IB) is a major threat to the global poultry industry. Despite the availability of commercial vaccines, the IB epidemic has not been effectively controlled. The exploration of novel IBV vaccines may provide a new way to prevent and control IB. In this study, BLP-S1, a bacterium-like particle displaying the S1 subunit of infectious bronchitis virus (IBV), was constructed using the GEM-PA surface display system. The immunoprotective efficacy results showed that BLP-S1 can effectively induce specific IgG and sIgA immune responses, providing a protection rate of 90% against IBV infection in 14-day-old commercial chickens. These results suggest that BLP-S1 has potential for the development of novel vaccines with good immunogenicity and immunoprotection.

Isolation and Identification of Novel Highly Pathogenic Avian Influenza Virus (H5N8) Subclade 2.3.4.4b from Geese in Northeastern China.

H5N8, a highly pathogenic avian influenza, has become a new zoonotic threat in recent years. As of December 28, 2021, at least 3,206 H5N8 cases had been reported in wild birds and poultry worldwide. In January 2021, a novel virus strain named A/goose/China/1/2021 was isolated during an H5N8 goose influenza outbreak in northeastern China. The PB2, PB1, HA, and M genes of A/goose/China/1/2021 were highly identical to those of H5N8 strains emerging in Kazakhstan and Russia in Central Asia from August to September 2020, while the remaining four genes had the closest homology to those of H5N8 viruses isolated in South Korea in East Asia from November to December 2020. We thus speculate that A/goose/China/1/2021 is likely a reassortant virus that formed in the 2020 to 2021 influenza season and that the migratory birds via the two migration routes of Central Asia and East Asia-Australia may have played an essential role in the genetic reassortment of this virus. The phylogenetic analysis indicated that the HA genes of H5N8 viruses belonging to group II of subclade 2.3.4.4b, including A/goose/China/1/2021, may be derived from strains in Central Asia. Given the complex global spread of H5N8 virus, our study highlights the necessity to strengthen the function of the global surveillance network for H5N8 virus and to accelerate the pace of vaccine development to confront the current challenges posed by H5N8 virus of subclade 2.3.4.4. IMPORTANCE H5N8, a highly pathogenic avian influenza, not only has an impact on public health, but also has a huge negative impact on animal health, food safety, safety, and even on the local and international economy. The migratory wild birds play a vital role in the intercontinental transmission of H5N8 virus. It is urgent that we should strengthen the function of the global surveillance network for H5N8 virus and accelerate the pace of vaccine development to confront the current challenges posed by H5N8 virus of subclade 2.3.4.4.

Association between triglyceride-glucose index and arterial stiffness reflected by carotid pulse-wave velocity in stage 1 hypertension and individuals with normal/elevated blood pressure.

Evidence of the triglyceride-glucose (TyG) index as an independent predictor of arterial stiffness in stage 1 hypertension patients is scarce. This study aimed to explore the association between TyG index and arterial stiffness in this population. A total of 1041 individuals from 32 centers with normal/elevated blood pressure (BP, <130/80 mmHg; 345 men (33%); median age, 37 years) and 585 stage 1 hypertension patients (BP ≥130/80 and <140/90 mmHg; 305 men (52%); median age, 47 years) were prospectively enrolled. Arterial stiffness was determined by measuring carotid ultrafast pulse-wave velocity (ufPWV). TyG index was calculated as ln (fasting triglyceride (TG) × fasting blood glucose/2). Patients with a higher TyG index tended to have higher ufPWV. The TyG index was positively associated with ufPWV at the end of systole in stage 1 hypertension patients after adjusting for confounding factors (ß for per unit .48), and restricted cubic spline analysis confirmed a linear association. Subgroup analyses in terms of age, sex, and body mass index yielded similar results. However, no significant relationship was observed between the TyG index and ufPWV in the population with normal/elevated BP. The fully adjusted ß between ufPWV and the TyG index was higher than the TG/high-density lipoprotein cholesterol ratio, TG, and pulse pressure. In conclusion, patients with a higher TyG index had greater arterial stiffness, and the TyG index independently and positively correlated with arterial stiffness in stage 1 hypertension patients. The TyG index may provide a simple and reliable marker to monitor arterial stiffness in hypertensive patients.

Oral administration of Lactobacillus plantarum JC7 alleviates OVA-induced murine food allergy through immunoregulation and restoring disordered intestinal microbiota.

PURPOSE: The incidence and prevalence of food allergy have sharply risen over the past several decades. Oral administration of probiotic stains has been proven as a safe and effective method to control food allergy. In this study, it aims to comprehensively investigate the anti-allergic effect of Lactobacillus plantarum JC7. METHODS: Balb/c mice were randomly divided into three groups and received OVA (20 µg/mouse, intraperitoneal injection), L. plantarum JC7 (2 × 108 CFU/mouse, intragastric administration) + OVA (20 µg/mouse, intraperitoneal injection) or 0.9% saline (intragastric administration) for 3 weeks. Body weight was monitored weekly, and allergic reactions were evaluated after challenge of OVA. Serum levels of OVA-specific immunoglobulins and various cytokines were tested using ELISA, and the cecum microbiota was analysed by 16S rRNA sequencing to explore the relationships between these indicators and OVA-induced food allergy. Western blotting was used to identify the expression levels of phosphorylated IκBα and nuclear factor kappa B p65. RESULTS: OVA-sensitised mice showed mitigation of respiratory manifestations, alleviation of lung inflammation and congestion, and the presence of an intact intestinal villus structure. Furthermore, OVA-specific immunoglobulin E (IgE), OVA-specific-IgG1, and plasma histamine levels were declined in mice treated with L. plantarum JC7 than in OVA-sensitised mice. In addition, interferon-γ (IFN-γ) and interleukin 10 (IL-10) levels were significantly increased, while IL-4 and IL-17A levels were clearly decreased in mice that had undergone oral administration of L. plantarum JC7, compared with OVA-sensitised mice. These findings indicated imbalances of T helper cell type 1 (Th1)/Th2 and regulatory T cells (Treg)/Th17, which were confirmed by quantitative polymerase chain reaction (PCR). Western blotting demonstrated that the expression levels of phosphorylated IκBα and nuclear factor kappa B p65 were significantly increased in OVA-sensitised mice, but these changes were partly reversed after treatment with L. plantarum JC7. Oral administration of L. plantarum JC7 increased the richness, diversity, and evenness of cecum microbiota, characterised by higher Bacteroidetes abundance and lower Firmicutes abundance. Additionally, the intestinal microbial community composition was significantly altered in the OVA-sensitised group, indicating a disordered intestinal microbiota that was restored by the oral administration of L. plantarum JC7. CONCLUSION: Overall, L. plantarum JC7 can prevent food allergy by rectifying Th1/Th2 and Treg/Th17 imbalances, combined with modifications of disordered intestinal microbiota.

A Single Vaccination of Chimeric Bivalent Virus-Like Particle Vaccine Confers Protection Against H9N2 and H3N2 Avian Influenza in Commercial Broilers and Allows a Strategy of Differentiating Infected from Vaccinated Animals.

H9N2 and H3N2 are the two most important subtypes of low pathogenic avian influenza viruses (LPAIV) because of their ongoing threat to the global poultry industry and public health. Although commercially available inactivated H9N2 vaccines are widely used in the affected countries, endemic H9N2 avian influenza remains uncontrolled. In addition, there is no available avian H3N2 vaccine. Influenza virus-like particles (VLPs) are one of the most promising vaccine alternatives to traditional egg-based vaccines. In this study, to increase the immunogenic content of VLPs to reduce production costs, we developed chimeric bivalent VLPs (cbVLPs) co-displaying hemagglutinin (HA) and neuraminidase (NA) of H9N2 and H3N2 viruses with the Gag protein of bovine immunodeficiency virus (BIV) as the inner core using the Bac-to-Bac baculovirus expression system. The results showed that a single immunization of chickens with 40µg/0.3mL cbVLPs elicited an effective immune response and provided complete protection against H9N2 and H3N2 viruses. More importantly, cbVLPs with accompanying serological assays can successfully accomplish the strategy of differentiating infected animals from vaccinated animals (DIVA), making virus surveillance easier. Therefore, this cbVLP vaccine candidate would be a promising alternative to conventional vaccines, showing great potential for commercial development.

A SYBR Green-based real-time RT-PCR assay to differentiate the H1N1 influenza virus lineages.

The H1N1 subtype influenza viruses (H1N1) have been causing persistent epidemics in human, swine and poultry populations since 1918. This subtype has evolved into four relatively stable genetic lineages, including classical swine influenza virus lineage, seasonal human influenza virus lineage, avian influenza virus lineage and Eurasian avian-like swine influenza virus lineage. In this study, four pairs of primers, based on the relatively conserved HA nucleotide regions of each H1N1 genetic lineage, were designed to establish a SYBR Green-based real-time quantitative RT-PCR (qPCR) assay to differentiate between the H1N1 genetic lineages. The results of qPCR assay showed that the lineage-specific primers designed for each H1N1 lineage were intra-lineage-specific, without mismatch of inter-lineage or inter-subtype and there appeared specific amplification curves when the concentrations of H1N1 plasmids were greater than or equal to 1.0 × 101 copies/reaction. Thus, this qPCR assay can specifically differentiate between the four lineages of H1N1 with a good specificity and sensitivity, which would assist in recognizing the infection and epidemic status of different H1N1 genetic lineages.

Preparation and evaluation of virus-like particle vaccine against H3N8 subtype equine influenza.

To prevent and control H3N8 subtype equine influenza, we prepared virus-like particles (VLPs) comprising the HA, NA and M1 proteins of H3N8 equine influenza virus (EIV) through the insect cell-baculovirus expression system. The results of Western blot and hemagglutination analyses demonstrated that the constructed VLPs comprising HA, NA and M1 proteins have good hemagglutination activity. Immunoelectron microscope revealed that the VLPs share similar morphology and structure with natural virus particles. The hyperimmune serum from horses immunized with the VLPs were injected into mice by means of artificial passive immunization and then challenge, or challenge following by injecting hyperimmune serum. The results showed that the equine hyperimmune serum has good preventive and therapeutic efficacy against the infection of H3N8 EIV. The study provides a technical foundation for the development of H3N8 EIV VLP vaccine.

Assessing the effects of a two-amino acid flexibility in the Hemagglutinin 220-loop receptor-binding domain on the fitness of Influenza A(H9N2) viruses.

The enzootic and zoonotic nature of H9N2 avian influenza viruses poses a persistent threat to the global poultry industry and public health. In particular, the emerging sublineage h9.4.2.5 of H9N2 viruses has drawn great attention. In this study, we determined the effects of the flexibility at residues 226 and 227 in the hemagglutinin on the receptor avidity and immune evasion of H9N2 viruses. The solid-phase direct binding assay showed that residue 226 plays a core role in the receptor preference of H9N2 viruses, while residue 227 affects the preference of the virus for a receptor. Consequently, each of these two successive residues can modulate the receptor avidity of H9N2 viruses and influence their potential of zoonotic infection. The antigenic map based on the cross-hemagglutination inhibition (HI) titers revealed that amino acid substitutions at positions 226 or 227 appear to be involved in antigenic drift, potentially resulting in the emergence of H9N2 immune evasion mutants. Further analysis suggested that increased receptor avidity facilitated by residue 226Q or 227M resulted in a reduction in the HI titer. Among the four naturally-occurring amino acid combinations comprising QQ, MQ, LQ, and LM, the number of viruses with LM accounted for 79.64% of the sublineage h9.4.2.5 and the rescued virus with LM exhibited absolute advantages of in vitro and in vivo replication and transmission. Collectively, these data demonstrate that residues 226 and 227 are under selective pressure and their synergistic regulation of receptor avidity and antigenicity is related to the evolution of circulating H9N2 viruses.

Reference Values of Carotid Ultrafast Pulse-Wave Velocity: A Prospective, Multicenter, Population-Based Study.

BACKGROUND: Ultrafast ultrasound imaging has been demonstrated to be an effective method to evaluate carotid stiffness through carotid pulse-wave velocity (PWV) with high reproducibility, but a lack of reference values has precluded its widespread use in clinical practice. The aims of this study were to establish reference values of PWV for ultrafast ultrasound imaging in a prospective, multicenter, population-based cohort study and to investigate the main determinants of carotid PWV. METHODS: A total of 1,544 healthy Han Chinese volunteers (581 men [38%]; age range, 18-95 years) were enrolled from 32 collaborating laboratories in China. The participants were categorized by age, blood pressure (BP), and body mass index (BMI). Basic clinical parameters and carotid PWV at the beginning of systole (BS) and at end-systole (ES) were measured using ultrafast ultrasound imaging techniques. RESULTS: PWV at both BS and ES was significantly higher in the left carotid artery than in the right carotid artery. PWV at BS was significantly higher in men than in women; however, no significant difference was noted in PWV at ES between men and women. Multiple linear regression analyses revealed that age, BP, and BMI were independently correlated with PWV at both BS and ES. PWV at BS and ES progressively increased with increases in age, BP, and BMI. Furthermore, age- and sex-specific reference values of carotid PWV for ultrafast ultrasound imaging were established. CONCLUSIONS: Reference values of carotid PWV for ultrafast ultrasound imaging, stratified by sex and age, were determined for the first time. Age, BP, and BMI were the dominant determinants of carotid PWV for ultrafast ultrasound imaging, which should be considered in clinical practice for assessing arterial stiffness.

Increased carotid stiffness detected by ultrafast ultrasound imaging is associated with the Gensini score.

AIM: To test the ability of carotid stiffness evaluated by using ultrafast ultrasound imaging to indicate coronary atherosclerosis and its association with the severity of coronary artery disease (CAD). MATERIAL AND METHODS: This cross-sectional study included 131 patients with CAD and 60 normal controls. Carotid intima-media thickness (cIMT) was measured by two-dimensional ultrasound. Carotid stiffness was determined by ultrafast ultrasound imaging, with which the carotid pulse wave velocity at the beginning (PWVBS) and end (PWVES) of systole were calculated. Gensini scores based on coronary angiography were used to estimate the severity of CAD. RESULTS: Compared with normal controls, the CAD patients had higher carotid diameters, cIMT, PWVBS and PWVES (p < 0.05). In the CAD group, Gensini scores correlated significantly with cIMT, PWVBS and PWVES (r = 0.279, p = 0.001; r = 0.661, p < 0.001; r = 0.620, p < 0.001; respectively). The multivariate analysis further indicated that PWVBS, PWVES and body mass index were independently associated with the Gensini score (ß = 0.466, p < 0.001; ß = 0.308, p < 0.001; and ß = 0.209, p = 0.001; respectively). In addition, the sensitivity and specificity were 54% and 83%, respectively, for PWVBS (cutoff value, 6.9 m/s; area under the receiver operating characteristic curve, 0.70) and 64% and 83%, respectively, for PWVES (cutoff value, 8.0 m/s; area under the curve, 0.73). CONCLUSIONS: Increased carotid PWVBS and PWVES detected by ultrafast ultrasound imaging as indices of carotid stiffness might serve as promising indicators for CAD and its severity.

Characterization of Newcastle disease virus obtained from toco toucan.

Given that the current Newcastle disease virus (NDV) infection in wild birds poses the threat to poultry, surveillance of Newcastle disease in captive wild birds was carried out in Jilin, China in 2018. Here, an NDV strain obtained from toco toucan was firstly characterized. The results showed that the F gene of the NDV isolate Toucan/China/3/2018 is classified as genotype II in class II. Sequence analysis of the F0 cleavage site was 113RQGR/L117, which supports the result of the intracerebral pathogenicity index assay indicating classification of the isolate as low-pathogenicity. Experimental infection demonstrated that Toucan/China/3/2018 can effectively replicate and transmit among chickens. To our knowledge, this is the first report on genetically and pathogenically characterizing NDV strain isolated from toucan, which enriches the epidemiological information of NDV in wild birds.

Endothelial function and plasma matrix metalloproteinase-2 levels and their association with the size and elastic properties of the ascending aorta in first-degree relatives of bicuspid aortic valve patients.

PURPOSE: To explore endothelial function and plasma matrix metalloproteinase-2 levels and their association with the size and elastic properties of the ascending aorta in the first-degree relatives (FDRs) with tricuspid aortic valve (TAV) of individuals with bicuspid aortic valve (BAV). METHODS: Twenty-six patients with BAV without significant valvular dysfunction, 35 FDRs with TAV, and 29 matched healthy controls were analyzed. Two-dimensional echocardiography and high-resolution ultrasound were applied to evaluate the size and elasticity of the ascending aorta and the flow-mediated vasodilation (FMD) of the brachial artery in response to hyperemia. RESULTS: The aortic diameter was larger in the BAV patients than in the FDRs and controls, and the aortic elastic properties showed larger decreases in the BAV patients. The FDRs had a larger aortic diameter and more impaired elastic properties than the controls. The BAV patients had a significantly lower FMD than the FDRs and controls (P < .001), while the FMD was lower in the FDRs than in the controls (P < .001). The plasma MMP-2 levels were significantly higher in the BAV patients than in the FDRs (P = .001) and controls (P < .001). Additionally, the FDRs had higher plasma MMP-2 levels than the controls (P < .001). Pearson correlation analysis revealed that the aortic diameter, distensibility, stiffness index, and strain were significantly associated with FMD and plasma MMP-2 levels. CONCLUSION: Aortic enlargement and impaired elasticity in the FDRs with TAV were associated with impaired endothelial function and increased plasma MMP-2 concentrations.