Carbonylation of Runx2 at K176 by 4-Hydroxynonenal Accelerates Vascular Calcification.

BACKGROUND: Vascular calcification, which is characterized by calcium deposition in arterial walls and the osteochondrogenic differentiation of vascular smooth muscle cells, is an actively regulated process that involves complex mechanisms. Vascular calcification is associated with increased cardiovascular adverse events. The role of 4-hydroxynonenal (4-HNE), which is the most abundant stable product of lipid peroxidation, in vascular calcification has been poorly investigated. METHODS: Serum was collected from patients with chronic kidney disease and controls, and the levels of 4-HNE and 8-iso-prostaglandin F2α were measured. Sections of coronary atherosclerotic plaques from donors were immunostained to analyze calcium deposition and 4-HNE. A total of 658 patients with coronary artery disease who received coronary computed tomography angiography were recruited to analyze the relationship between coronary calcification and the rs671 mutation in aldehyde dehydrogenase 2 (ALDH2). ALDH2 knockout (ALDH2-/-) mice, smooth muscle cell-specific ALDH2 knockout mice, ALDH2 transgenic mice, and their controls were used to establish vascular calcification models. Primary mouse aortic smooth muscle cells and human aortic smooth muscle cells were exposed to medium containing ß-glycerophosphate and CaCl2 to investigate cell calcification and the underlying molecular mechanisms. RESULTS: Elevated 4-HNE levels were observed in the serum of patients with chronic kidney disease and model mice and were detected in calcified artery sections by immunostaining. ALDH2 knockout or smooth muscle cell-specific ALDH2 knockout accelerated the development of vascular calcification in model mice, whereas overexpression or activation prevented mouse vascular calcification and the osteochondrogenic differentiation of vascular smooth muscle cells. In patients with coronary artery disease, patients with ALDH2 rs671 gene mutation developed more severe coronary calcification. 4-HNE promoted calcification of both mouse aortic smooth muscle cells and human aortic smooth muscle cells and their osteochondrogenic differentiation in vitro. 4-HNE increased the level of Runx2 (runt-related transcription factor-2), and the effect of 4-HNE on promoting vascular smooth muscle cell calcification was ablated when Runx2 was knocked down. Mutation of Runx2 at lysine 176 reduced its carbonylation and eliminated the 4-HNE-induced upregulation of Runx2. CONCLUSIONS: Our results suggest that 4-HNE increases Runx2 stabilization by directly carbonylating its K176 site and promotes vascular calcification. ALDH2 might be a potential target for the treatment of vascular calcification.

Occurrence, removal, emission and environment risk of 32 antibiotics and metabolites in wastewater treatment plants in Wuhu, China.

Wastewater treatment plants (WWTPs) are considered important sources of antibiotics and metabolites in aquatic environments and pose a serious threat to the safety of aquatic organisms. In this study, we investigated the seasonal occurrence, removal, emission, and environmental risk assessment (ERA) of 32 antibiotics and metabolites at four WWTPs located in Wuhu, China. The main findings of this study are as follows: Ofloxacin concentrations dominated all WWTPs, and large quantities of sulfachinoxalin were only detected in WWTP 2 treating mixed sewage. The average apparent removal of individual parent antibiotics or metabolites ranged from -94.7 to 100 %. There was a noticeable seasonal emission pattern (independent t-test, t = 9.89, p < 0.001), with lower emissions observed during summer. WWTPs discharged 85.2 ± 43.8 g of antibiotics and metabolites each day. Approximately 87 % of emissions were discharged into the mainstream of the Yangtze River, while the remainder were discharged into its tributary, the Zhanghe River. The total emissions of 21 parent antibiotics were approximately 18 % of the prescription data, indicating that a considerable and alarming amount of prototype drugs entered the receiving water body. Based on the risk quotient (RQ) of the ERA, the Zhanghe River has a moderate risk of ofloxacin (RQ = 0.111-0.583), a low or insignificant risk of sulfamethoxazole (RQ = 0.003-0.048), and an insignificant risk of other antibiotics or metabolites. However, the risk of antibiotics or metabolites in the mainstream of Yangtze River is insignificant. This study could help understand the seasonal emission patterns of antibiotics and metabolites, as well as more antibiotics sensitive of environmental risks in tributary than that in mainstream.

VEGF Mediates Tumor Growth and Metastasis by Affecting the Expression of E-Cadherin and N-Cadherin Promoting Epithelial to Mesenchymal Transition in Gastric Cancer.

Background: Gastric cancer (GC) is the fifth leading cancer in the world, and there is a high mortality rate in China. Exploring the relationship between the prognosis of GC and the expression of related genes is helpful to further understand the common characteristics of the occurrence and development of GC and provide a new method for the identification of early GC, so as to provide the best therapeutic targets. Methods: Vascular endothelial growth factor (VEGF) and markers of epithelial-mesenchymal transition (EMT) were investigated immunohistochemically using tumor samples obtained from 196 GC tissues and adjacent tumor tissues. The correlation of the expression level with histopathologic features and survival was investigated. Results: Here, we show that VEGF and EMT markers expression were significantly correlated with depth of tumor invasion and GC stage (P < .05), degree of differentiation and lymph node metastasis (P < .001). We found that the rate of VEGF positivity in GC tissues was 52.05%, which was significantly higher than that in adjacent cancer tissues (16.84%). In GC, the association between VEGF and E-cadherin was negative (r = -0.188, P < .05), whereas VEGF and N-cadherin were positively correlated (r = 0.214, P < .05). Furthermore, the Kaplan-Meier analysis and a Cox regression model were used to analyze the effect of VEGF and EMT marker expression on the survival of the patients. We found that the overall survival of GC patients was correlated with VEGF (P < .001), N-cadherin (P < .001), E-cadherin (P = .002) expression, and some histopathologic features. Conclusions: Vascular endothelial growth factor and EMT markers exist side by side and play a part together in the development of GC, which provides new ideas for evaluating the prognosis of GC and researching targeted drugs.

Cross-species infection potential of avian influenza H13 viruses isolated from wild aquatic birds to poultry and mammals.

Wild aquatic birds are the primary hosts of H13 avian influenza viruses (AIVs). Herein, we performed a genetic analysis of two H13 AIVs isolated from wild birds in China and evaluated their infection potential in poultry to further explore the potential for transmission from wild aquatic birds to poultry. Our results showed that the two strains belong to different groups, one strain (A/mallard/Dalian/DZ-137/2013; abbreviated as DZ137) belongs to Group I, whereas the other strain (A/Eurasian Curlew/Liaoning/ZH-385/2014; abbreviated as ZH385) belongs to Group III. In vitro experiments showed that both DZ137 and ZH385 can replicate efficiently in chicken embryo fibroblast cells. We found that these H13 AIVs can also efficiently replicate in mammalian cell lines, including human embryonic kidney cells and Madin-Darby canine kidney cells. In vivo experiments showed that DZ137 and ZH385 can infect 1-day-old specific pathogen-free (SPF) chickens, and that ZH385 has a higher replication ability in chickens than DZ137. Notably, only ZH385 can replicate efficiently in 10-day-old SPF chickens. However, neither DZ137 nor ZH385 can replicate well in turkeys and quails. Both DZ137 and ZH385 can replicate in 3-week-old mice. Serological surveillance of poultry showed a 4.6%-10.4% (15/328-34/328) antibody-positive rate against H13 AIVs in farm chickens. Our findings indicate that H13 AIVs have the replication ability in chickens and mice and may have a risk of crossing the host barrier from wild aquatic birds to poultry or mammals in the future.

The NO-cGMP-PKG Axis in HFpEF: From Pathological Mechanisms to Potential Therapies.

Heart failure with preserved ejection fraction (HFpEF) accounts for almost half of all heart failure (HF) cases worldwide. Unfortunately, its incidence is expected to continue to rise, and effective therapy to improve clinical outcomes is lacking. Numerous efforts currently directed towards the pathophysiology of human HFpEF are uncovering signal transduction pathways and novel therapeutic targets. The nitric oxide-cyclic guanosine phosphate-protein kinase G (NO-cGMP-PKG) axis has been described as an important regulator of cardiac function. Suppression of the NO-cGMP-PKG signalling pathway is involved in the progression of HFpEF. Therefore, the NO-cGMP-PKG signalling pathway is a potential therapeutic target for HFpEF. In this review, we aim to explore the mechanism of NO-cGMP-PKG in the progression of HFpEF and to summarize potential therapeutic drugs that target this signalling pathway.

Severe immunochemotherapy-induced toxicities in a patient with dyskeratosis congenita and literature review.

OBJECTIVES: Dyskeratosis congenita (DC) is a rare inherited disease characterized by the triad of reticulate hyperpigmentation, nail dystrophy and oral leukoplakia. DC patients are considered vulnerable to external pressure, such as immunochemotherapy. There are very few cases reporting severe therapy-induced toxicities in patients with DC. METHODS: A 27-year-old woman was admitted to our hospital with a 4-month history of pancytopenia and a 7-day history of dyspnea with coughing. She was diagnosed with non-Hodgkin's lymphoma 5 months ago. She received immunochemotherapy due to non-Hodgkin's lymphoma but experienced recurrent fever, oral ulcer, pancytopenia, dyspnea and other symptoms during immunochemotherapy. On admission, she experienced an aggravation of respiratory symptoms, recurrent infections and acute heart failure. RESULTS: Laboratory examination confirmed pancytopenia, and chest computed tomography showed interstitial lung disease (ILD). Genetic analysis results confirmed the presence of DC and a TINF2 gene mutation. With continuous supportive and anti-infection treatment, her condition finally stabilized. She was discharged from the hospital after nearly 2 months. DISCUSSION: We reviewed similar cases and found common features that could be useful. However, the reported cases are very limited. More cases and studies are needed. CONCLUSION: These cases indicate that DC patients seem more vulnerable to therapy toxicities; thus, physicians should be careful when treating these patients with chemotherapy drugs or radiation therapy. Reduced-intensity therapy may be considered.

PLXDC1 Can Be a Biomarker for Poor Prognosis and Immune Evasion in Gastric Cancer.

Background: Research has revealed that Plexin domain containing 1 (PLXDC1) is correlated with the prognosis of a variety of tumors, but its role in the tumor microenvironment (TME) of gastric cancer has not been reported. Methods: In this study, we analyzed PLXDC1 expression in gastric cancer using the Oncomine and the Cancer Genome Atlas (TCGA) databases and immunohistochemical staining experiments, and performed prognostic assessment with data from the TCGA and Kaplan-Meier Plotter databases. The immunomodulatory role of PLXDC1 in the gastric cancer TME was analyzed by signaling pathway enrichment, immune cell correlation analysis, immunomodulator risk model construction and immunohistochemical staining experiments of immune cells. Results: The results indicated that PLXDC1 was overexpressed in gastric cancer and that its overexpression was associated with poor prognosis. Multivariate Cox analysis revealed that PLXDC1 could be an independent biomarker of the risk of gastric cancer. Signaling pathway enrichment revealed that high PLXDC1 expression was involved in signaling pathways related to immune activation and stromal activation, and Tumor Immune Dysfunction and Exclusion (TIDE) assessment indicated that high PLXDC1 expression was associated with a significantly higher risk of immune evasion than low PLXDC1 expression. A Cox risk model based on PLXDC1-associated immunomodulators also presented poor prognosis, and immune evasion was significantly higher in the high-risk group than in the low-risk group. In addition, immunohistochemical staining of CD8/CD3/CD4+ T cells in the high and low PLXDC1 expression groups also observed immune cell distribution characteristics of immune evasion. Conclusion: This study analyzed PLXDC1 from multiple biological perspectives and revealed that PLXDC1 can be a biomarker for poor prognosis and immune evasion in gastric cancer.

The upregulation of stromal antigen 3 expression suppresses the phenotypic hallmarks of hepatocellular carcinoma through the Smad3-CDK4/CDK6-cyclin D1 and CXCR4/RhoA pathways.

BACKGROUND: The stromal antigen 3 (STAG3) gene encodes an adhesion complex subunit that can regulate sister chromatid cohesion during cell division. Chromosome instability caused by STAG3 gene mutation may potentially promote tumor progression, but the effect of STAG3 on hepatocellular carcinoma (HCC) and the related molecular mechanism are not reported in the literature. The mechanism of the occurrence and development of HCC is not adequately understood. Therefore, the biological role of STAG3 in HCC remains to be studied, and whether STAG3 might be a sensitive therapeutic target in HCC remains to be determined. METHODS: The expression and clinical significance of STAG3 in HCC tissues and cell lines were determined by RT-qPCR and immunohistochemistry analyses. The biological functions of STAG3 in HCC were determined through in vitro and in vivo cell function tests. The molecular mechanism of STAG3 in HCC cells was then investigated by western blot assay. RESULTS: The mRNA expression of STAG3 was lower in most HCC cells than in normal cells. Subsequently, an immunohistochemical analysis of STAG3 was performed with 126 samples, and lower STAG3 expression was associated with worse overall survival in HCC patients. Moreover, cytofunctional tests revealed that the lentivirus-mediated overexpression of STAG3 in HCC cells inhibited cell proliferation, migration, and invasion; promoted apoptosis; induced G1/S phase arrest in vitro; and inhibited tumor growth in vivo. Furthermore, studies of the molecular mechanism suggested that the overexpression of STAG3 increased Smad3 expression and decreased CDK4, CDK6, cyclin D1, CXCR4 and RhoA expression. CONCLUSION: STAG3 exhibits anticancer effects against HCC, and these effects involve the Smad3-CDK4/CDK6-cyclin D1 and CXCR4/RhoA pathways. STAG3 is a tumor-suppressor gene that may serve as a potential target for molecular therapy, which provides a new idea for the treatment of HCC.

Peri-operative Takotsubo syndrome after non-cardiac surgery: a retrospective nested case-control study.

AIMS: Takotsubo syndrome (TTS) is an acute reversible cardiac dysfunction that may occur during the peri-operative period and among patients with serious illness. We aimed to evaluate the clinical characteristics, peri-operative management, and prognosis of peri-operative TTS (pTTS) and explore the factors associated with pTTS. METHODS: We conducted a retrospective nested case-control study using the database of patients who underwent in-hospital non-cardiac surgeries between January 2017 and December 2020 in Peking University Third hospital. Cases were adult patients diagnosed TTS at discharge who were matched with four controls based on operative types. Multivariable conditional logistic regression was used to identified the factors associated with pTTS. The area under the curve (AUC) was used to evaluate the diagnostic efficacy. RESULTS: Among the 128 536 patients underwent non-cardiac surgery, 20 patients with pTTS and 80 patients without were enrolled in this study. The incidence of pTTS was about 0.016% in our centre. The median age of patients with pTTS was 52.5 (38.25, 76.25) years, although 90% of them were female. Fifty per cent (9 cases) of female patients were pre-menopausal. Caesarean section has the highest proportion of pTTS (30% of the pTTS cases) with the incidence of caesarean section-related pTTS of 0.06% in our centre. A high prevalence of non-apical ballooning pattern of regional wall motion abnormality (seven cases, 35%) and a high mortality (two cases, 10%) were observed. Left ventricular ejection fraction (LVEF) of patients with pTTS was significantly decreased (41.7 ± 8.8%). In the acute phase, supportive treatments aiming to reduce life-threatening complications were main treatment strategies. After receiving systematic treatment, significant improvements were observed in LVEF (63.1 ± 13.5%), with median recovery time of LVEF of 7.48 days. Leucocyte count [odds ratio (OR): 4.59; 95% confidence interval (CI): 1.10-19.15], haemoglobin (HGB) (OR: 10.52; 95% CI: 1.04-106.36), and the revised cardiac risk index (RCRI) score (OR: 6.30; 95% CI: 1.05-37.88) were the factors significantly associated with pTTS. The RCRI score performed poorly in the prediction of pTTS (AUC: 0.630; 95% CI: 0.525-0.735). After adding leucocyte count and HGB into the RCRI score, the AUC was significantly improved (AUC: 0.768; 95% CI: 0.671-0.865; P = 0.001). CONCLUSIONS: Patients with pTTS have some differences compared with common TTS, including higher proportion of pre-menopausal female, higher prevalence during caesarean section, higher prevalence of non-apical ballooning pattern of regional wall motion abnormality, and higher mortality. The RCRI score performed poorly in the evaluation of pTTS. Adding HGB and leucocyte count into the RCRI score could significantly improve its predictive performance.

ANKFN1 plays both protumorigenic and metastatic roles in hepatocellular carcinoma.

Ankyrin repeat and fibronectin type III domain containing 1 (ANKFN1) is reported to be involved in human height and developmental abnormalities, but the expression profile and molecular function of ANKFN1 in hepatocellular carcinoma (HCC) remain unknown. This study aimed to evaluate the clinical significance and biological function of ANKFN1 in HCC and investigate whether ANKFN1 can be used for differential diagnosis in HCC. Here, we showed that ANKFN1 was upregulated in 126 tumor tissues compared with adjacent nontumorous tissues in HCC patients. The upregulation of ANKFN1 in HCC was associated with cirrhosis, alpha-fetoprotein (AFP) levels and poor prognosis. Moreover, silencing ANKFN1 expression suppressed HCC cell proliferation, migration, invasion, and metastasis in vitro and subcutaneous tumorigenesis in vivo. However, ANKFN1 overexpression promoted HCC proliferation and metastasis in an orthotopic liver transplantation model and attenuated the above biological effects in HCC cells. ANKFN1 significantly affected HCC cell proliferation by inducing G1/S transition and cell apoptosis. Mechanistically, we demonstrated that ANKFN1 promoted cell proliferation, migration, and invasion via activation of the cyclin D1/Cdk4/Cdk6 pathway by stimulating the MEK1/2-ERK1/2 pathway. Moreover, ANKFN1-induced cell proliferation, migration, and invasion were partially reversed by ERK1/2 inhibitors. Taken together, our results indicate that ANKFN1 promotes HCC cell proliferation and metastasis by activating the MEK1/2-ERK1/2 signaling pathway. Our work also suggests that ANKFN1 is a potential therapeutic target for HCC.

Malignant Peripheral Nerve Sheath Tumor of the Cervix.

   The occurrence of primary malignant peripheral neurilemmoma in the female cervix without neurofibromatosis type 1 is extremely rare. We, herein, report a case of a 46-year woman with malignant peripheral nerve sheath tumor (MPNST) involving the cervix. The patient was admitted to the hospital because of irregular vaginal bleeding. Pelvic CT suggested the presence of cervical mass, and the patient accepted radical hysterectomy. Following the surgery, MPNST was diagnosed by a combination of histology and immunohistochemistry. One year later, the patient developed recurrence and pulmonary metastasis and received sequentially epiubicin/ifosfamide, dacarbazine, and etoposide/cisplatin multi-line chemotherapy. The prognosis of MPNST is poor, which is related to its invasiveness, high recurrence and metastasis rates. The patient was treated with surgery, multi-line chemotherapy and targeted therapy, but the overall survival (OS) was 44 months. It is the first reported case of cervical MPNST receiving combined treatment. Key Words: Malignant peripheral nerve sheath tumor, Cervix, Pulmonary metastasis, Hysterectomy, Chemotherapy.

CHSY3 can be a Poor Prognostic Biomarker and Mediates Immune Evasion in Stomach Adenocarcinoma.

Background: Chondroitin sulphate synthase 3 (CHSY3) is an important enzyme that regulates glycosylation, but it has not been reported in tumours. This study explored for the first time the oncological features of CHSY3 in stomach adenocarcinoma (STAD). Methods: We analysed CHSY3 expression in STAD through the Cancer Genome Atlas (TCGA) database and verified our findings by immunohistochemical staining and Western blot experiments. The prognostic value of CHSY3 in STAD was analysed through the biological aspects of CHSY3 in STAD, such as communal clinical follow-up survival data, methylation sites, tumour immune microenvironment (TIME) and immune cell surface checkpoints. Finally, the immune-evasion potential of CHSY3 in STAD was assessed on the Tumor Immune Dysfunction and Exclusion (TIDE) website and immunohistochemical staining experiment. Results: CHSY3 overexpression in STAD was associated with a poor prognosis based on immunohistochemical staining and Western blot experiments. Multivariate Cox analysis suggested that CHSY3 could be an independent prognostic risk factor. Pathway enrichment and TIME analysis demonstrated that CHSY3 up-regulated mesenchymal activation and immune activation signals in STAD, while TIDE assessment revealed that the risk of immune evasion was significantly higher in the high CHSY3 expression group than in the low CHSY3 expression group. Risk model scores based on CHSY3-associated immune cell surface checkpoints also presented poor prognosis, and immune evasion was significantly higher in the high-risk group than in the low-risk group. Conclusions: This study analysed CHSY3 from multiple biological perspectives and revealed that CHSY3 can be a biomarker of poor prognosis and mediates the TIME immune-evasion status in STAD.

Development and Validation of Predictive Model-HASBLAD Score-For Major Adverse Cardiovascular Events During Perioperative Period of Non-cardiac Surgery: A Single Center Experience in China.

Background: Major adverse cardiovascular events (MACEs) represent a significant reason of morbidity and mortality in non-cardiac surgery during perioperative period. The prevention of perioperative MACEs has always been one of the hotspots in the research field. Current existing models have not been validated in Chinese population, and have become increasingly unable to adapt to current clinical needs. Objectives: To establish and validate several simple bedside tools for predicting MACEs during perioperative period of non-cardiac surgery in Chinese hospitalized patients. Design: We used a nested case-control study to establish our prediction models. A nomogram along with a risk score were developed using logistic regression analysis. An internal cohort was used to evaluate the performance of discrimination and calibration of these predictive models including the revised cardiac risk index (RCRI) score recommended by current guidelines. Setting: Peking University Third Hospital between January 2010 and December 2020. Patients: Two hundred and fifty three patients with MACEs and 1,012 patients without were included in the training set from January 2010 to December 2019 while 38,897 patients were included in the validation set from January 2020 and December 2020, of whom 112 patients had MACEs. Main Outcome Measures: The MACEs included the composite outcomes of cardiac death, non-fatal myocardial infarction, non-fatal congestive cardiac failure or hemodynamically significant ventricular arrhythmia, and Takotsubo cardiomyopathy. Results: Seven predictors, including Hemoglobin, CARDIAC diseases, Aspartate aminotransferase (AST), high Blood pressure, Leukocyte count, general Anesthesia, and Diabetes mellitus (HASBLAD), were selected in the final model. The nomogram and HASBLAD score all achieved satisfactory prediction performance in the training set (C statistic, 0.781 vs. 0.768) and the validation set (C statistic, 0.865 vs. 0.843). Good calibration was observed for the probability of MACEs in the training set and the validation set. The two predictive models both had excellent discrimination that performed better than RCRI in the validation set (C statistic, 0.660, P < 0.05 vs. nomogram and HASBLAD score). Conclusion: The nomogram and HASBLAD score could be useful bedside tools for predicting perioperative MACEs of non-cardiac surgery in Chinese hospitalized patients.

Short-term exposure to traffic-related air pollution and STEMI events: Insights into STEMI onset and related cardiac impairment.

AIMS: Evidence on the impacts of traffic-related air pollution (TRAP) on ST-segment elevation myocardial infarction (STEMI) events is limited. We aimed to assess the acute effects of TRAP exposure on the clinical onset of STEMI and related cardiac impairments. METHODS AND RESULTS: We recruited patients who were admitted for STEMI and underwent primary percutaneous coronary intervention at Peking University Third Hospital between 2014 and 2020. Indicators relevant to cardiac impairments were measured. Concomitantly, hourly concentrations of traffic pollutants were monitored throughout the study period, including fine particulate matter, black carbon (BC), particles in size ranges of 5-560 nm, oxides of nitrogen (NOX), nitrogen dioxide, and carbon monoxide. The mean (SD) age of participants was 62.4 (12.5) years. Daily average (range) concentrations of ambient BC and NOX were 3.9 (0.1-25.0) µg/m3 and 90.8 (16.6-371.7) µg/m3. Significant increases in STEMI risks of 5.9% (95% CI: 0.1, 12.0) to 21.9% (95% CI: 6.0, 40.2) were associated with interquartile range increases in exposure to TRAP within a few hours. These changes were accompanied by significant elevations in cardiac troponin T levels of 6.9% (95% CI: 0.2, 14.1) to 41.7% (95% CI: 21.2, 65.6), as well as reductions in left ventricular ejection fraction of 1.5% (95% CI: 0.1, 2.9) to 3.7% (95% CI: 0.8, 6.4). Furthermore, the associations were attenuated in participants living in areas with higher residential greenness levels. CONCLUSIONS: Our findings extend current understanding that short-term exposure to higher levels of traffic pollution was associated with increased STEMI risks and exacerbated cardiac impairments, and provide evidence on traffic pollution control priority for protecting vulnerable populations who are at greater risks of cardiovascular events.

H9N2 influenza virus spillover into wild birds from poultry in China bind to human-type receptors and transmit in mammals via respiratory droplets.

H9N2 influenza virus has been reported worldwide for several decades, and it has evolved into multiple genotypes among domestic poultry. However, the study involving ecology and evolution of low pathogenic avian influenza virus H9N2 in wild birds in China is limited. Here, we carried out surveillance of avian influenza virus H9N2 in wild birds along with the East Asian-Australian migratory flyway in China in 2017. To estimate the prevalence of H9N2 avian virus in wild birds, information on exposure of wild bird populations to H9N2 viruses using serology, in addition to virology, would greatly improve monitoring capabilities. In this study, we also present serological data of H9N2 among wild birds in China during 2013-2016. We report the identification of poultry-derived H9N2 isolates from asymptomatic infected multispecies wild birds such as Common kestrel (Falco tinnunculus), Northern goshawk (Accipiter gentilis), Little owl (Athene noctua) and Ring-necked Pheasant (Phasianus colchicus) in North China in June 2017. Phylogenetic analysis demonstrated that Tianjin H9N2 isolates belong to the G81 and carry internal genes highly homologous to human H10N8 and H7N9. The isolates could directly infect mice without adaptation but were restricted to replicate in the respiratory system. Glycan-binding preference analyses suggested that the H9N2 isolates have acquired a binding affinity for the human-like receptor. Notably, results from transmission experiment in guinea pigs and ferrets demonstrated the wild birds-derived H9N2 influenza virus exhibits efficient transmission phenotypes in mammalian models via respiratory droplets. Our results indicate that the H9N2 AIVs continued to circulate extensively in wild bird populations and migratory birds play an important role in the spread and genetic diversification of H9N2 AIVs. The pandemic potential of H9N2 viruses demonstrated by aerosol transmission in mammalian models via respiratory droplets highlights the importance of monitoring influenza viruses in these hosts.

Safety, Immunogenicity, and Protective Efficacy of an H5N1 Chimeric Cold-Adapted Attenuated Virus Vaccine in a Mouse Model.

H5N1 influenza virus is a threat to public health worldwide. The virus can cause severe morbidity and mortality in humans. We constructed an H5N1 influenza candidate virus vaccine from the A/chicken/Guizhou/1153/2016 strain that was recommended by the World Health Organization. In this study, we designed an H5N1 chimeric influenza A/B vaccine based on a cold-adapted (ca) influenza B virus B/Vienna/1/99 backbone. We modified the ectodomain of H5N1 hemagglutinin (HA) protein, while retaining the packaging signals of influenza B virus, and then rescued a chimeric cold-adapted H5N1 candidate influenza vaccine through a reverse genetic system. The chimeric H5N1 vaccine replicated well in eggs and the Madin-Darby Canine Kidney cells. It maintained a temperature-sensitive and cold-adapted phenotype. The H5N1 vaccine was attenuated in mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H5N1 HA protein. The chimeric H5N1 candidate vaccine protected mice against lethal challenge with a wild-type highly pathogenic avian H5N1 influenza virus. The chimeric H5 candidate vaccine is thus a potentially safe, attenuated, and reassortment-incompetent vaccine with circulating A viruses.

Amino acid sites related to the PB2 subunits of IDV affect polymerase activity.

BACKGROUND: In 2011, a new influenza virus, named Influenza D Virus (IDV), was isolated from pigs, and then cattle, presenting influenza-like symptoms. IDV is one of the causative agents of Bovine Respiratory Disease (BRD), which causes high morbidity and mortality in feedlot cattle worldwide. To date, the molecular mechanisms of IDV pathogenicity are unknown. Recent IDV outbreaks in cattle, along with serological and genetic evidence of IDV infection in humans, have raised concerns regarding the zoonotic potential of this virus. Influenza virus polymerase is a determining factor of viral pathogenicity to mammals. METHODS: Here we take a prospective approach to this question by creating a random mutation library about PB2 subunit of the IDV viral polymerase to test which amino acid point mutations will increase viral polymerase activity, leading to increased pathogenicity of the virus. RESULTS: Our work shows some exact sites that could affect polymerase activities in influenza D viruses. For example, two single-site mutations, PB2-D533S and PB2-G603Y, can independently increase polymerase activity. The PB2-D533S mutation alone can increase the polymerase activity by 9.92 times, while the PB2-G603Y mutation increments the activity by 8.22 times. CONCLUSION: Taken together, our findings provide important insight into IDV replication fitness mediated by the PB2 protein, increasing our understanding of IDV replication and pathogenicity and facilitating future studies.