Phosphorylation of PB2 at serine 181 restricts viral replication and virulence of the highly pathogenic H5N1 avian influenza virus in mice.

Influenza A virus (IAV) continues to pose a pandemic threat to public health, resulting a high mortality rate annually and during pandemic years. Posttranslational modification of viral protein plays a substantial role in regulating IAV infection. Here, based on immunoprecipitation (IP)-based mass spectrometry (MS) and purified virus-coupled MS, a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified, including 60 novel phosphorylation sites. Additionally, for the first time, we provide evidence that PB2 can also be acetylated at site K187. Notably, the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS. Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains, suggesting their fundamental importance in the IAV life cycle. Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure. While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice, PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice. Mechanistically, PB2 S181E substantially impairs viral polymerase activity and viral replication, remarkably dampens protein stability and nuclear accumulation of PB2, and significantly weakens IAV-induced inflammatory responses. Therefore, our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins, laying a foundation for subsequent mechanistic studies. Meanwhile, the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.

Blood urea nitrogen to serum albumin ratio: a good predictor of in-hospital and 90-day all-cause mortality in patients with acute exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Previous studies on acute exacerbation of chronic obstructive pulmonary disease (AECOPD) have found that those who died in hospital had higher blood urea nitrogen levels and a worse nutritional status compared to survivors. However, the association between the blood urea nitrogen to serum albumin ratio (BUN/ALB ratio) and in-hospital and short-term prognosis in patients with AECOPD remains unclear. The aim of this study was to explore the usefulness of BUN/ALB ratio in AECOPD as an objective predictor for in-hospital and 90-day all-cause mortality. METHODS: We recorded the laboratory and clinical data in patients with AECOPD on admission. By drawing the ROC curve for the patients, we obtained the cut-off point for the BUN/ALB ratio for in-hospital death. Multivariate logistic regression was used for analyses of the factors of in-hospital mortality and multivariate Cox regression was used to analyze the factors of 90-day all-cause mortality. RESULTS: A total of 362 patients were recruited and 319 patients were finally analyzed. Twenty-three patients died during hospitalization and the fatality rate was 7.2%. Furthermore, 14 patients died by the 90-day follow-up. Compared with in-hospital survivors, patients who died in hospital were older (80.78 ± 6.58 vs. 75.09 ± 9.73 years old, P = 0.001), had a higher prevalence of congestive heart failure(69.6% vs. 27.4%, P < 0.001), had a higher BUN/ALB ratio [0.329 (0.250-0.399) vs. 0.145 (0.111-0.210), P < 0.001], had higher neutrophil counts [10.27 (7.21-14.04) vs. 6.58 (4.58-9.04), P < 0.001], higher blood urea nitrogen levels [10.86 (7.10-12.25) vs. 5.35 (4.14-7.40), P < 0.001], a lower albumin level (32.58 ± 3.72 vs. 36.26 ± 4.53, P < 0.001) and a lower lymphocyte count [0.85 (0.58-1.21) vs. 1.22 (0.86-1.72), P = 0.001]. The ROC curve showed that the area under the curve (AUC) of BUN/ALB ratio for in-hospital death was 0.87, (95%CI 0.81-0.93, P < 0.001), the best cut-off point value to discriminate survivors from non-survivors in hospital was 0.249, the sensitivity was 78.3%, the specificity was 86.5%, and Youden's index was 0.648. Having a BUN/ALB ratio ≥ 0.249 was an independent risk factor for both in-hospital and 90-day all-cause mortality after adjustment for relative risk (RR; RR = 15.08, 95% CI 3.80-59.78, P < 0.001 for a multivariate logistic regression analysis) and hazard ratio (HR; HR = 5.34, 95% CI 1.62-17.57, P = 0.006 for a multivariate Cox regression analysis). CONCLUSION: An elevated BUN/ALB ratio was a strong and independent predictor of in-hospital and 90-day all-cause mortality in patients with AECOPD.

Prognostic Role of Chronic Obstructive Pulmonary Disease and Asthma Physiology Score for in-Hospital and 1-year Mortality in Patients with Acute Exacerbations of COPD.

BACKGROUND AND OBJECTIVES: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) often lead to high mortality. Chronic obstructive pulmonary disease and asthma physiology score (CAPS) is a simple clinical severity score. The aim of this study was to explore whether CAPS could be an effective predictor for in-hospital and 1-year mortality in AECOPD patients. Methods. We used CAPS to grade all patients and record their clinical characteristics. The receiver operator characteristic (ROC) curve was used to determine the cut-off of CAPS that discriminated survivors and non-survivors. Univariate and multivariate logistic regression analyses and Cox regression analyses were used to identify the risk factors for in-hospital and 1-year mortality, respectively. Results. 240 patients were enrolled in our study; 18 patients died during hospitalization and 29 patients died during the 1-year follow-up. Compared with in-hospital survivors, those who died were older (80.83 ± 6.06 vs. 76.94 ± 8.30 years old, P = 0.019) and had a higher percentage of congestive heart failure (61.1% vs. 14.4%, P < 0.001), higher CAPS levels (31.11 ± 10.05 vs. 16.49 ± 7.11 points, P < 0.001), and a lower BMI (19.48 ± 3.26 vs. 21.50 ± 3.86, P = 0.032). The area under the ROC curve of CAPS for in-hospital death was 0.91 (95% CI: 0.85-0.96) with a sensitivity of 0.889 and a specificity of 0.802 for a cut-off point of 21 points. CAPS ≥21 points was an independent risk factor for in-hospital mortality after adjustment for relative risk (RR) (RR = 13.28, 95% CI: 1.97-89.53, P = 0.008). Univariate Cox regression analysis showed that a CAPS ≥21 points (HR = 4.07, 95% CI: 1.97-8.44) was a risk factor for 1-year mortality. However, multivariate Cox regression analysis showed that CAPS (HR = 2.24, 95% CI: 0.90-5.53) was not associated with 1-year mortality. CONCLUSION: A CAPS ≥21 points was a strong and independent risk factor for in-hospital mortality in AECOPD patients and CAPS had no impact on the 1-year mortality in patients with acute exacerbations of COPD after discharge.

Association Between Extracellular Superoxide Dismutase Activity and 1-Year All-Cause Mortality in Patients With Acute Exacerbations of Chronic Obstructive Pulmonary Disease: A Prospective Cohort Study.

Background and Objectives: Accumulating evidence suggests that oxidative stress is involved in the development of chronic obstructive pulmonary disease (COPD) and its progression. Activity of extracellular superoxide dismutase (ecSOD), the only extracellular enzyme eliminating superoxide radicals, has been reported to decline in acute exacerbations of COPD (AECOPD). However, the association between serum ecSOD activity and 1-year all-cause mortality in AECOPD patients remains unclear. The objective of our study was to explore the usefulness of ecSOD activity on admission in AECOPD as an objective predictor for 1-year all-cause mortality. Methods: We measured serum ecSOD activity in AECOPD patients on admission in a prospective cohort study. We also recorded their laboratory and clinical data. Multivariate Cox regression was used to analyze the association between ecSOD activity and the risk of 1-year all-cause mortality. Restricted cubic spline curves were used to visualize the relationship between ecSOD activity and the hazard ratio of 1-year all-cause mortality. Results: A total of 367 patients were followed up for 1 year, and 29 patients died during a 1-year follow-up period. Compared with survivors, the non-survivors were older (79.52 ± 8.39 vs. 74.38 ± 9.34 years old, p = 0.004) and had increased levels of tobacco consumption (47.07 ± 41.67 vs. 33.83 ± 31.79 pack-years, p = 0.037). Having an ecSOD activity ≤ 98.8 U/ml was an independent risk factor of 1-year all-cause mortality after adjustment for baseline differences, clinical variables and comorbidities [hazard ratio = 5.51, 95% confidence interval (CI): 2.35-12.95, p < 0.001]. Conclusion: Lower serum ecSOD activity was a strong and independent predictor of 1-year all-cause mortality in AECOPD patients.

Characterization of two chicken origin highly pathogenic H7N9 viruses isolated in northern China.

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attention should be paid to the importance of continual surveillance of the H7N9 AIVs.

Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay.

Swine influenza is an economically important respiratory disease in swine, but it also constantly poses a threat to human health. Therefore, developing rapid, sensitive, and efficient detection methods for swine influenza virus (SIV) is important. By aligning the haemagglutinin (HA) gene sequences of SIVs circulating in China over a 10-year period, an H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and pandemic 2009 H1N1 ((H1N1)pdm09) lineages plus a H3 primer-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed. Subsequently, a TaqMan-MGB-based duplex one-step real-time RT-PCR (RT-qPCR) assay was established and evaluated. The duplex RT-qPCR has a detection limit of 5 copies/µL of HA plasmid for EA H1N1, (H1N1)pdm09, and HL H3N2 subtype SIVs, and its overall detection sensitivity of 100% and specificity of 91.67% matches that of traditional virus isolation through chicken embryo inoculation using experimentally infected mouse lung samples. The method showed high repeatability both within run and between runs, and there was no cross-reactivity against several other porcine viruses that are commonly circulating in China. Furthermore, the duplex RT-qPCR method revealed a higher prevalence of subtype H1 than subtype H3 in 166 nasal swabs from pigs collected from one slaughterhouse between October and December 2019. This assay could be very helpful in the rapid differential detection and routine surveillance of EA H1N1, (H1N1)pdm09, and HL H3N2 SIVs in China.

The virulence modulator PA-X protein has minor effect on the pathogenicity of the highly pathogenic H7N9 avian influenza virus in mice.

PA-X is a novel discovered accessory protein encoded by the PA mRNA of the influenza A virus. Accumulated studies have demonstrated the crucial role of this protein in regulating the virulence of various subtypes of influenza virus, including H1N1, H5N1, H9N2, H1N2, H3N8 and H3N2 virus. However, the role of PA-X protein in regulating the virulence of the highly pathogenic avian H7N9 virus was unknown. In this study, we firstly generated two recombinant H7N9 viruses which have lower PA-X expression level than the parental H7N9 virus. We then systematically compared their difference in virus replication, polymerase activity, virulence and virus-induced host immune responses in mice. The results showed that the PA-X deficient viruses significantly increased viral replication in madin darby canine kidney cells and slightly increased viral replication in mouse lung. In addition, loss of PA-X expression significantly increased viral polymerase activity and alleviated the host-shutoff activity mediated by the parental PA protein. However, in contrast with the usual function of PA-X in regulating the virulence in different subtype influenza virus, no obvious effect on viral virulence in mice was observed by H7N9 PA-X protein. Furthermore, among the 12 kinds of cytokines and 2 kinds of complement derived components that we tested, the PA-X deficiency viruses only induced significantly higher expression levels of MX1 than the parental virus. Altogether, these results showed that PA-X has little effect on viral virulence and viral induced innate immune response of the H7N9 subtype virus. Our study adds further information for the growing understanding of the complexity of PA-X in regulating viral virulence and host innate immune response of different influenza virus.

Mutations during the adaptation of H7N9 avian influenza virus to mice lungs enhance human-like sialic acid binding activity and virulence in mice.

The first avian H7N9 influenza outbreak in spring of 2013 emerged in an unprecedented transmission from infected poultry to humans in the Yangtze delta area, eastern China, posing a dual challenge to public health and poultry industry. However, the mechanism for how avian H7N9 influenza virus adapts to mammalian hosts has not been clearly understood. Here, to identify adaptive changes that confer enhanced virulence of H7N9 virus in mammals, we generated a mouse-adapted H7N9 variant virus (S8) by serial lung-to-lung passages of the wild-type SDL124 virus in mice and compared their phenotype in vivo and in vitro. Sequence analysis showed that the two viruses differed by 27 amino acids distributed among six genes, containing changes in PB2 (E627K, D701N) and HA (Q226L) genes. The 50% mouse lethal dose (MLD50) of S8 reduced about 500 folds, to be moderately pathogenic to mice when compared to that of low pathogenic wild-type SDL124. Moreover, S8 replicated efficiently in mouse lungs and displayed expanded tissue tropism, and induced a greater degree of pulmonary edema and higher level of inflammatory cell infiltration in bronchoalveolar lavage fluids than SDL124 did. Interestingly, the mouse adapted S8 virus obtained strong affinity for human-like (SAα-2,6 Gal) receptor during the adaptation in mice. Correspondingly, compared with SDL124 virus, S8 virus showed higher replication efficiency in mammalian cells, whereas lower replication ability in avian cells. Taken together, these findings suggest that these mutations synergistically elevate the ability of H7N9 virus to disseminate to multiple organs and subsequently enhance the virulence of H7N9 virus in mammalian hosts.

Prognostic Role of NT-proBNP for in-Hospital and 1-Year Mortality in Patients with Acute Exacerbations of COPD.

Background and objective: The association between N-terminal pro B-type natriuretic peptide (NT-proBNP) concentrations and in-hospital and 1-year mortality in acute exacerbations of chronic obstructive pulmonary disease (AECOPD) patients is largely unknown. Our objective was to explore the usefulness of NT-proBNP concentrations in AECOPD patients as a prognostic marker for in-hospital and 1-year mortality. Methods: NT-proBNP levels were measured in patients upon admission and laboratory and clinical data were also recorded. The cut-point for the NT-proBNP concentration level for in-hospital death was obtained using the receiver operating characteristic (ROC) curve. Univariate and multivariate logistic regression and Cox regression were used in the analyses of factors of in-hospital and 1-year mortality. Results: A total of 429 patients were enrolled. Twenty-nine patients died during hospitalization and 59 patients died during the 1-year follow-up. Patients who died in-hospital compared with those in-hospital survivors were older (80.14±6.56 vs 75.93±9.45 years, p=0.003), had a higher percentage of congestive heart failure (65.52% vs 33.75%, p<0.001), had higher NT-proBNP levels (5767.00 (1372.50-12,887.00) vs 236.25 (80.03-1074.75) ng/L, p<0.001), higher neutrophil counts (10.52±5.82 vs 7.70±4.31, p=0.016), higher D-dimer levels (1231.62±1921.29 vs 490.11±830.19, p=0.048), higher blood urea nitrogen levels (9.91±6.33 vs 6.51±4.01 mmol/L, p=0.001), a lower body mass index (19.49±3.57 vs 22.19±4.76, p=0.003), and higher hemoglobin levels (122.34±25.36 vs 130.57±19.63, p=0.034). The area under the ROC curve (AUC) for NT-proBNP concentration was 0.88 (95% confidence interval [CI], 0.84-0.93). NT-proBNP concentrations ≥551.35 ng/L were an independent prognostic factor for both in-hospital and 1-year mortality after adjustment for relative risk (RR) (RR=29.54, 95% CI 3.04-286.63, p=0.004 for the multivariate logistic regression analysis) and hazard ratio (HR) (HR=4.47, 95% CI, 2.38-8.41, p <0.001 for the multivariate cox regression analysis). Conclusion: NT-proBNP was a strong and independent predictor of in-hospital and 1-year mortality in AECOPD patients.