Mutations in Polymerase Genes Enhanced the Virulence of 2009 Pandemic H1N1 Influenza Virus in Mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis

A Systematic Molecular Pathology Study of a Laboratory Confirmed H5N1 Human Case

Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo

A Comparison of Etiology, Pathogenesis, Vaccinal and Antiviral Drug Development between Influenza and COVID-19

Influenza virus and coronavirus, two kinds of pathogens that exist widely in nature, are common emerging pathogens that cause respiratory tract infections in humans. In December 2019, a novel coronavirus SARS-CoV-2 emerged, causing a severe respiratory infection named COVID-19 in humans, and raising a global pandemic which has persisted in the world for almost three years. Influenza virus, a seasonally circulating respiratory pathogen, has caused four global pandemics in humans since 1918 by the emergence of novel variants. Studies have shown that there are certain similarities in transmission mode and pathogenesis between influenza and COVID-19, and vaccination and antiviral drugs are considered to have positive roles as well as several limitations in the prevention and control of both diseases. Comparative understandings would be helpful to the prevention and control of these diseases. Here, we review the study progress in the etiology, pathogenesis, vaccine and antiviral drug development for the two diseases

Image_1_Deficiency of C-reactive protein or human C-reactive protein transgenic treatment aggravates influenza A infection in mice.jpeg

C-reactive protein (CRP) has been shown to be a potential candidate target in the immunotherapy of severe influenza A infection. However, it is unclear on the pathogenesis associated with CRP in influenza infections. Here, we used influenza A H1N1 CA04 to infect human CRP transgenic mice (KI), CRP knockout mice (KO), and wild-type mice (WT), respectively, and compared the viral pathogenicity and associated immune response in those mice. The results showed that CA04 infection resulted in 100%, 80%, and 60% death in KO, KI, and WT mice, respectively. Compared to WT mice, CA04 infection resulted in higher TCID50 in lungs on day 3 after infection but lowered HI antibody titers in sera of survivors on day 21 after infection in KI mice. ELISA assay showed that IFN-γ concentration was significantly increased in sera of WT, KI, or KO mice on day 7 after infection, and IL-17 was remarkably increased in sera of WT mice but decreased in sera of KI mice while no significant change in sera of KO mice on day 3 or 7 after infection. Quantitative RT-PCR showed that the relative expression levels of immune checkpoint CTLA-4, LAIR-1, GITR, BTLA, TIM-3, or PD-1 mRNA in the lung presented decreased levels on day 3 or 7 after infection in KI or KO mice. The correlation analysis showed that mRNA expression levels of the 6 molecules positively correlated with viral TICD50 in WT mice but negatively correlated with viral TCID50 in KI or KO mice. However, only LAIR-1 presented a significant correlation in each lung tissue of WT, KI, or KO mice with CA07 infection statistically. IHC results showed that LAIR-1 positive cells could be found in WT, KO, or KI mice lung tissues with CA04 infection, and the positive cells were mainly distributed in an inflammatory dense area. Our results suggested that deficiency of CRP or human CRP transgenic treatment aggravates influenza A virus infection in mice. CRP is a double sword in immune regulation of influenza infection in which IL-17 and immune checkpoint may be involved.</p

Development and implementation of the quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1) surveillance network in mainland China

Abstract Background Reverse transcription PCR (RT-PCR) and real time RT-PCR (rRT-PCR) have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B), serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA) and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1) surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1), can be obtained relatively easily through implementation of the panel on networks.</p

Database search for SC_PB2-H357N or PA-A36T mutation in virus isolates from nature.

<p>Polymorphisms of PA-36 and PB2-357 in influenza virus isolates were assessed and compared with our findings. The data shown are the number of sequences of different influenza subtypes in the NCBI database, the number of sequences of identical same amino acid composition as SC_WT and SC_M, and the number of sequences with a different amino acid at the same position (with the amino acids shown in parentheses). SC_M, SC_PB2-H357N/PA-A36T; Others*, subtypes which were not listed above; —, not applicable.</p

Growth properties of recombinant viruses in human (A, B), porcine (C, D) and murine cells (E, F).

<p>Confluent monolayer of A549, PK15 and LA-4 cell lines were inoculated with SC_WT, SC_PA-A36T or SC_PB2-H357N virus at MOI of 0.0001. Culture supernatants were harvested at 12, 24, 48, 60, 72 and 96 hpi at 35(<b>A, C, E</b>) or 39°C (<b>B, D, F</b>), respectively. Virus titers were determined by TCID₅₀ assay using MDCK cells. Results are presented as mean ± SEM and are representative of three determinations. *, °, <i>p</i><0.05, when comparing SC_PA-A36T and SC_PB2-H357N with SC_WT respectively, as determined by a <i>t</i>-test of TCID₅₀ values. **, °°, <i>p</i><0.001, as determined by <i>t</i>-test.</p

Viral RNA polymerase activity of SC_WT, SC_PA-A36T and SC_PB2-H357N in 293T cells cultured at different temperatures.

<p>Luciferase-based minigenome reporter assays were used to measure polymerase activity in 293T cells at 33, 37, or 39°C. Cells were co-transfected with Gluc reporter plasmid and expression plasmids PB1 and NP, PA, and PB2 (WT or PA-A36T, PB2-H357N mutants) to generate different viral RNPs. After culturing at 33, 37, or 39°C for 24 h, <i>Gaussia</i> luciferase production was measured. Results are presented as mean ± SEM and are representative of three determinations. **, <i>p</i><0.001, as determined by <i>t</i>-test.</p

Metabolic profiles in community-acquired pneumonia: developing assessment tools for disease severity

Abstract Background This study aimed to determine whether community-acquired pneumonia (CAP) had a metabolic profile and whether this profile can be used for disease severity assessment. Methods A total of 175 individuals including 119 CAP patients and 56 controls were enrolled and divided into two cohorts. Serum samples from a discovery cohort (n = 102, including 38 non-severe CAP, 30 severe CAP, and 34 age and sex-matched controls) were determined by untargeted ultra-high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics. Selected differential metabolites between CAP patients versus controls, and between the severe CAP group versus non-severe CAP group, were confirmed by targeted mass spectrometry assays in a validation cohort (n = 73, including 32 non-severe CAP, 19 severe CAP and 22 controls). Pearson’s correlation analysis was performed to assess relationships between the identified metabolites and clinical severity of CAP. The area under the curve (AUC), sensitivity and specificity of the metabolites for predicting the severity of CAP were also investigated. Results The metabolic signature was markedly different between CAP patients and controls. Fifteen metabolites were found to be significantly dysregulated in CAP patients, which were mainly mapped to the metabolic pathways of sphingolipid, arginine, pyruvate and inositol phosphate. The alternation trends of five metabolites among the three groups including sphinganine, p-Cresol sulfate, dehydroepiandrosterone sulfate (DHEA-S), lactate and l-arginine in the validation cohort were consistent with those in the discovery cohort. Significantly lower concentrations of sphinganine, p-Cresol sulfate and DHEA-S were observed in CAP patients than in controls (p  65 years (CURB-65), pneumonia severity index (PSI) and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, while DHEA-S inversely correlated with the three scoring systems. Combining lactate, sphinganine and DHEA-S as a metabolite panel for discriminating severe CAP from non-severe CAP exhibited a better AUC of 0.911 (95% confidence interval 0.825–0.998) than CURB-65, PSI and APACHE II scores. Conclusions This study demonstrates that serum metabolomics approaches based on the LC-MS/MS platform can be applied as a tool to reveal metabolic changes during CAP and establish a metabolite signature related to disease severity. Trial registration ClinicalTrials.gov, NCT03093220. Registered retrospectively on 28 March 2017