Organotypic lung tissue culture as a preclinical model to study host- influenza A viral infection: A case for repurposing of nafamostat mesylate.

Reliable and effective models for recapitulation of host-pathogen interactions are imperative for the discovery of potential therapeutics. Ex vivo models can fulfill these requirements as the multicellular native environment in the tissue is preserved and be utilized for toxicology, vaccine, infection and drug efficacy studies due to the presence of immune cells. Drug repurposing involves the identification of new applications for already approved drugs that are not related to the prime medical indication and emerged as a strategy to cope with slow pace of drug discovery due to high costs and necessary phases to reach the patients. Within the scope of the study, broad-spectrum serine protease inhibitor nafamostat mesylate was repurposed to inhibit influenza A infection and evaluated by a translational ex vivo organotypic model, in which human organ-level responses can be achieved in preclinical safety studies of potential antiviral agents, along with in in vitro lung airway culture. The safe doses were determined as 10 µM for in vitro, whereas 22 µM for ex vivo to be applied for evaluation of host-pathogen interactions, which reduced virus infectivity, increased cell/tissue viability, and protected total protein content by reducing cell death with the inflammatory response. When the gene expression levels of specific pro-inflammatory, anti-inflammatory and cell surface markers involved in antiviral responses were examined, the significant inflammatory response represented by highly elevated mRNA gene expression levels of cytokines and chemokines combined with CDH5 downregulated by 5.1-fold supported the antiviral efficacy of NM and usability of ex vivo model as a preclinical infection model.

Long-term outcomes for epidemic viral pneumonia survivors after discharge from the intensive care unit: a systematic review

ABSTRACT Objective To review the long-term outcomes (functional status and psychological sequelae) of survivors of critical illnesses due to epidemic viral pneumonia before the COVID-19 pandemic and to establish a benchmark for comparison of the COVID-19 long-term outcomes. Methods This systematic review of clinical studies reported the long-term outcomes in adults admitted to intensive care units who were diagnosed with viral epidemic pneumonia. An electronic search was performed using databases: MEDLINE®, Web of Science™, LILACS/IBECS, and EMBASE. Additionally, complementary searches were conducted on the reference lists of eligible studies. The quality of the studies was assessed using the Newcastle-Ottawa Scale. The results were grouped into tables and textual descriptions. Results The final analysis included 15 studies from a total of 243 studies. This review included 771 patients with Influenza A, Middle East Respiratory Syndrome, and Severe Acute Respiratory Syndrome. It analyzed the quality of life, functionality, lung function, mortality, rate of return to work, rehospitalization, and psychiatric symptoms. The follow-up periods ranged from 1 to 144 months. We found that the quality of life, functional capacity, and pulmonary function were below expected standards. Conclusion This review revealed great heterogeneity between studies attributed to different scales, follow-up time points, and methodologies. However, this systematic review identified negative long-term effects on patient outcomes. Given the possibility of future pandemics, it is essential to identify the long-term effects of viral pneumonia outbreaks. This review was not funded. Prospero database registration: (www.crd.york.ac.uk/prospero) under registration ID CRD42021190296.

Effect and Mechanism of Menispermi Rhizoma Total Alkaloids on Antiviral Infection / 中国实验方剂学杂志

ObjectiveTo investigate the antiviral effect of Menispermi Rhizoma total alkaloids and its relationship with the type Ⅰ interferon （IFN-Ⅰ） signaling pathway. MethodThe effects of Menispermi Rhizoma total alkaloids on the intracellular replication of influenza A virus （H1N1）， vesicular stomatitis virus （VSV）， and cerebral myocarditis virus （EMCV） were detected by fluorescent inverted microscope， flow cytometry， Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot. A mouse model infected with H1N1 was constructed， and the mice were divided into a control group， H1N1 model group， Menispermi Rhizoma total alkaloids groups （10， 20， 30 mg·kg-1）， and oseltamivir group （40 mg·kg-1）， so as to study the effects on the weight and survival rate of infected mice. Real-time PCR was used to detect the activation effect of Menispermi Rhizoma total alkaloids on the IFN-Ⅰ pathway in cells， and the relationship between the antiviral effect of Menispermi Rhizoma total alkaloids in IFNAR1 knockout A549 cells （IFNAR1-/--A549） and IFN-Ⅰ pathway was detected. ResultCompared with the control group， the virus proliferated significantly in the model group （P<0.01）. Compared with the model group， Menispermi Rhizoma total alkaloids could significantly inhibit the replication of H1N1， VSV， and EMCV in vitro （P<0.01）， inhibit the weight loss of the mice infected with the H1N1 in vivo， and improve the survival rate of mice （P<0.05）. In addition， Menispermi Rhizoma total alkaloids activated the IFN-I pathway and relied on this pathway to exert the function of antiviral infection. ConclusionMenispermi Rhizoma total alkaloids exert antiviral effects in vivo and in vitro by activating the IFN-Ⅰ pathway.

Biological, social, and healthcare factors associated with death due to influenza A(H1N1) during the 2009 epidemic in Brazil

Background: In June 2009, the World Health Organization (WHO) raised the global alert level for the A(H1N1)pdm09 influenza pandemic and at that time sustained transmission in Brazil was established. It was urgent to carry out studies that evaluated possible risk factors for death from Influenza A(H1N1) to improve case management strategies to reduce the lethality of the disease. This study aimed to identify risk factors for death from Influenza A(H1N1), including the effectiveness of the vaccine against influenza A(H1N1) concerning mortality. Methods: A case-control of incident cases of influenza A(H1N1) reported in the Epidemiological Information Systems of the states of São Paulo, Paraná, Pará, Amazonas, and Rio Grande do Sul was conducted. Results: 305 participants were included, 70 of them cases and 235 controls, distributed as follows: Amazonas ­ 9 cases/10 controls, Pará ­ 22 cases/77 controls, São Paulo ­ 19 cases/49 controls, Paraná ­ 10 cases/54 controls, Rio Grande do Sul ­ 10 cases/45 controls. These participants had a mean age of 30 years, with 33 years among cases and 25 years among controls. There was a predominance of females both among cases and controls. Biological (age), pre-existing diseases (congestive heart failure, respiratory disease, and diabetes mellitus), and care factors (ICU admission) associated with death from Influenza A(H1N1) were identified. Conclusion: The risk factors identified in this investigation allowed subsidizing the elaboration of clinical conducts, but also indicate important aspects for facing "new" influenza epidemics that are likely to occur in our country.

A dual fragment triggered DNA ladder nanostructure based on logic gate and dispersion-to-localization catalytic hairpin assembly for efficient fluorescence assay of SARS-CoV-2 and H1N1.

DNA nanotechnology has been widely utilized in the construction of various functional nanostructures. However, most DNA nanostructures have the shortcomings of low response rate and serious background leakage. Herein, we proposed the conception of AND logic gate cascaded dispersion-to-localization catalytic hairpin assembly (AND gate-DLCHA) for the fabrication of novel DNA ladder nanostructures. In our design, the entropy-driven AND logic gate can precisely recognize two fragments of the target nucleic acid sequences. After AND logic gate activation by target nucleic acids, dispersion-to-localization catalytic hairpin assembly was initiated. Consequently, tremendous DNA ladder nanostructures were generated and the response signal was rapidly enhanced, which can be used for rapid and amplied detection of nucleic acids. Taking advantage of the sensitivity and specificity of AND gate-DLCHA strategy, the fluorescence sensors were established and successfully applied in ultrasensitive assay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (H1N1) within 45 min with the limit of detection (LOD) as low as 66 copies mL-1 (SARS-CoV-2) and 33 copies mL-1 (H1N1), which showed perspectives in pathogen identification and biomedical application. The high selectivity and reliability of established sensors was attributed to the dual-fragment analysis. Meanwhile, the sensors possessed minimal leakage and greatly enhanced signal to background (S/B) ratio owing to substrate transduction from dispersion into colocalization. This rationally developed logic gate cascaded dispersion-to-localization catalytic hairpin assembly strategy presented a new approach for the development of DNA nanostructures.

Inference of age-dependent case-fatality ratios for seasonal influenza virus subtypes A(H3N2) and A(H1N1)pdm09 and B lineages using data from the Netherlands.

Background: Despite the known relatively high disease burden of influenza, data are lacking regarding a critical epidemiological indicator, the case-fatality ratio. Our objective was to infer age-group and influenza (sub)type specific values by combining modelled estimates of symptomatic incidence and influenza-attributable mortality. Methods: The setting was the Netherlands, 2011/2012 through 2019/2020 seasons. Sentinel surveillance data from general practitioners and laboratory testing were synthesised to supply age-group specific estimates of incidence of symptomatic infection, and ecological additive modelling was used to estimate influenza-attributable deaths. These were combined in an Bayesian inferential framework to estimate case-fatality ratios for influenza A(H3N2), A(H1N1)pdm09 and influenza B, per 5-year age-group. Results: Case-fatality estimates were highest for influenza A(H3N2) followed by influenza B and then A(H1N1)pdm09 and were highest for the 85+ years age-group, at 4.76% (95% credible interval [CrI]: 4.52-5.01%) for A(H3N2), followed by influenza B at 4.08% (95% CrI: 3.77-4.39%) and A(H1N1)pdm09 at 2.51% (95% CrI: 2.09-2.94%). For 55-59 through 85+ years, the case-fatality risk was estimated to double with every 3.7 years of age. Conclusions: These estimated case-fatality ratios, per influenza sub(type) and per age-group, constitute valuable information for public health decision-making, for assessing the retrospective and prospective value of preventative interventions such as vaccination and for health economic evaluations.

The Immunosuppressive Roles of PD-L1 during Influenza A Virus Infection.

The clinical benefits of targeting programmed death-ligand 1 (PD-L1) in various cancers represent a strategy for the treatment of immunosuppressive diseases. Here, it was demonstrated that the expression levels of PD-L1 in cells were greatly upregulated in response to H1N1 influenza A virus (IAV) infection. Overexpression of PD-L1 promoted viral replication and downregulated type-I and type-III interferons and interferon-stimulated genes. Moreover, the association between PD-L1 and Src homology region-2, containing protein tyrosine phosphatase (SHP2), during IAV/H1N1 infection was analyzed by employing the SHP2 inhibitor (SHP099), siSHP2, and pNL-SHP2. The results showed that the expressions of PD-L1 mRNA and protein were decreased under SHP099 or siSHP2 treatment, whereas the cells overexpressing SHP2 exhibited the opposite effects. Additionally, the effects of PD-L1 on the expression of p-ERK and p-SHP2 were investigated in PD-L1-overexpressed cells following WSN or PR8 infection, determining that the PD-L1 overexpression led to the decreased expression of p-SHP2 and p-ERK induced by WSN or PR8 infection. Taken together, these data reveal that PD-L1 could play an important role in immunosuppression during IAV/H1N1 infection; thus, it may serve as a promising therapeutic target for development of novel anti-IAV drugs.

Acute necrotising encephalopathy: A rare complication providing a clue for the outbreak of H1N1 among medical students in Kanpur, India.

Kanpur, India, recently witnessed an outbreak of undifferentiated febrile illness among medical students. Several students developed high-grade fever with altered sensorium within 2-3 days after the index case. Surprisingly, this outbreak coincided with the death of several pigs in the vicinity. Acute necrotising encephalitis, although rare, was noted in some patients. When correlated with each other, all of these incidents were suggestive of an outbreak of H1N1.

In Vitro Anti-Influenza A Virus H1N1 Effect of Sesquiterpene-Rich Extracts of Carpesium abrotanoides.

Due to a high content of sesquiterpenes, Carpesium abrotanoides has been investigated to fully explore its health-promoting properties. Therefore, this work aimed to assess, for the first time, the anti-influenza A virus H1N1 potential of sesquiterpene-targeted fractions of the herb derived from C. abrotanoides. Five compounds, including four sesquiterpenes and one aldehyde, were isolated and identified from the sesquiterpene-rich extracts of C. abrotanoides (SECA), and the contents of three main sesquiterpenes in the SECA were determined. Furthermore, SECA showed a significant protective effect in the MDCK cells infected with influenza A virus (H1N1) in three different conditions: premixed administration, prophylactic administration, and therapeutic administration. SECA can significantly decrease the mRNA expressions of TLR4, MyD88, NF-κB, TNF-α, and IL-6, as well as the protein expressions of TLR4, MyD88, and NF-κB. This result suggests that SECA can resist the influenza A virus H1N1 through the TLR4/MyD88/NF-κB signal pathway.

Modulation of Immune Responses Against HA1 Influenza Vaccine Candidate by B-lymphocyte Stimulator Cytokine in Mice.

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied. The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 µg/µL of rHA1 were mixed with 2 µg/µL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain. Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose. The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection.

LC-QTOF-MS/MS Based Molecular Networking Approach for the Isolation of α-Glucosidase Inhibitors and Virucidal Agents from Coccinia grandis (L.) Voigt.

Coccinia grandis or ivy gourd is an edible plant. Its leaves and fruits are used as vegetable in many countries. Many works on antidiabetic activity of a crude extract of C. grandis, i.e., in vitro, in vivo, and clinical trials studies, have been reported. Profiles of the antidiabetic compounds were previously proposed by using LC-MS or GC-MS. However, the compounds responsible for antidiabetic activity have rarely been isolated and characterized by analysis of 1D and 2D NMR data. In the present work, UHPLC-ESI-QTOF-MS/MS analysis and GNPS molecular networking were used to guide the isolation of α-glucosidase inhibitors from an extract of C. grandis leaves. Seven flavonoid glycosides including rutin (1), kaempferol 3-O-rutinoside (2) or nicotiflorin, kaempferol 3-O-robinobioside (3), quercetin 3-O-robinobioside (4), quercetin 3-O-ß-D-apiofuranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-glucopyranoside (5) or CTN-986, kaempferol 3-O-ß-D-api-furanosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-glucopyranoside (6), and kaempferol 3-O-ß-D-apiofuranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-galactopyranoside (7) were isolated from C. grandis leaves. This is the first report of glycosides containing apiose sugar in the genus Coccinia. These glycosides exhibited remarkable α-glucosidase inhibitory activity, being 4.4-10.3 times more potent than acarbose. Moreover, they also displayed virucidal activity against influenza A virus H1N1, as revealed by the ASTM E1053-20 method.

Anti-bacterial and anti-viral effects of Fengreqing oral liquid () in vitro and in vivo.

OBJECTIVE: To investigate the anti-bacterial and anti-viral effects of Fengreqing oral liquid (, FRQ) in vitro and in vivo. METHODS: The minimum inhibitory concentrations of Fengreqing Oral Liquid against six gram-positive bacteria (Staphylococcus aureus, Streptococcus mutans, Peptostreptococcus anaerobius, Hemolytic streptococcus, Streptococcus pneumoniae, Klebsiella pneumoniae), seven gram-negative bacteria (Escherichia coli, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Haemophilus influenzae, Helicobacter pylori, Pseudomonas aeruginosa, Gardnerella vaginalis) and Candida albicans were detected by the paper disc diffusion method. The inhibition rate of A/PuertoRico/8/34(H1N1) (PR8) influenza virus in different concentrations of Fengreqing oral solution was detected by chicken embryo method. CCK8 method was used to detect the half-cell infection of RSV, VSV and CVB3. The effect of FRQ on the survival curve of mice was detected by using co-infection model of Streptococcus pneumoniae and influenza virus. RESULTS: In vitro, FRQ can inhibit Actinobacillus actinomycetemcomitans, Helicobacter pylori, Gardnerella vaginalis, Staphylococcus aureus, Streptococcus mutans and Streptococcus pneumoniae and has an antiviral effect on the envelope virus H1N1. In vivo, Fengreqing oral solution had therapeutic effect on influenza-Streptococcus pneumoniae co-infection in mice, significantly improving the survival rate of mice. The medium dose and low dose FRQ significantly prolonged the survival time of mice. CONCLUSION: FRQ has good anti-bacterial and anti-viral effectsin vivo and in vitro.

Comparison of two pandemics: H1N1 and SARS-CoV-2

SUMMARY OBJECTIVE: We aimed to compare the clinical, epidemiological, and prognostic features of the H1N1 pandemic in 2009 and the severe acute respiratory syndrome coronavirus 2 pandemic in 2020. METHODS: This retrospective study involved subjects from seven centers that were admitted and found to be positive for H1N1 or COVID-19 real-time polymerase chain reaction test. RESULTS: A total of 143 patients with H1N1 and 309 patients with COVID-19 were involved in the study. H1N1 patients were younger than COVID-19 ones. While 58.7% of H1N1 patients were female, 57.9% of COVID-19 patients were male. Complaints of fever, cough, sputum, sore throat, myalgia, weakness, headache, and shortness of breath in H1N1 patients were statistically higher than in COVID-19 ones. The duration of symptoms until H1N1 patients were admitted to the hospital was shorter than that for COVID-19 patients. Leukopenia was more common in COVID-19 patients. C-reactive protein levels were higher in COVID-19 patients, while lactate dehydrogenase levels were higher in H1N1 ones. The mortality rate was also higher in H1N1 cases. CONCLUSIONS: The severe acute respiratory syndrome coronavirus 2 pandemic is a major public health problem that continues to affect the world with its high rate of contagion. In addition, no vaccines or a specific drug for the benefit of millions of people have been found yet. The H1N1 pandemic is an epidemic that affected the whole world about ten years ago and was prevented by the development of vaccines at a short period. Experience in the H1N1 pandemic may be the guide to prevent the COVID-19 pandemic from a worse end.

Respiratory Viral Infections in Sickle Cell Anemia: Special Emphasis on H1N1 Co-infection.

OBJECTIVES: Patients with sickle cell anemia (SCA) are immunocompromised and at an increased risk of developing infections. Our aim was to establish the clinical, laboratory, and radiological manifestations of respiratory viral infections in SCA at Sultan Qaboos University Hospital (SQUH), Oman and assess its impact on disease morbidity and mortality, with special emphasis on H1N1. METHODS: We undertook a retrospective study in SCA patients with respiratory viral infections following up at the hematology department at SQUH. We collected demographic data and clinical, radiological, and laboratory parameters. RESULTS: In 84 SCA patients with 109 admission episodes for vaso-occlusive crisis (VOC), molecular diagnostic techniques confirmed 125 respiratory viral infections. Rhinovirus was the most prevalent infection (35.8%), whereas H1N1 virus infection was seen only in 10.1%. Laboratory investigations revealed a significant fall in mean hemoglobin levels, mean white blood cell, and platelet counts from baseline, whereas there was a significant rise in the mean lymphocyte and retic count, serum lactate dehydrogenase, and C-reactive protein levels during infective episodes (p < 0.050, Wilcoxon signed rank test). One-third (32.1%) of the VOC episodes progressed to acute chest syndrome (ACS), but in the H1N1 cohort, only two episodes of ACS was seen (18.2%). CONCLUSIONS: Rhinovirus was the commonest respiratory virus infections in SCA patients, whereas parainfluenza 3 was associated with a significant adverse outcome. H1N1 was associated with a mild course. ACS was seen in approximately one-third of this group of patients.

Reduning plus ribavirin display synergistic activity against severe pneumonia induced by H1N1 influenza A virus in mice.

OBJECTIVE: To investigate synergistic effect of Reduning (RDN) injection plus ribavirin against severe pneumonia induced by H1N1 influenza A virus in mice. METHODS: We established a mouse model of severe pneumonia induced by influenza A virus by infecting Balb/c mice with CA07 virus. We randomly assigned the infected mice into four groups, and treated them with normal saline (NS group), RDN (injection, 86.6 mg/kg), ribavirin (injection, 66.6 mg/kg) or double Ribavirin plus RDN group, the same dosage as used in the single treatments) for 5 d. Lung index and lung pathology were recorded or calculated in terms of the curative effective. Cytokines, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome related protein including caspase-associated recruitment domain (CARD) domain Apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC), caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3), and reactive oxygen species were simultaneously investigated. RESULTS: RDN plus ribavirin treatment, not RDN or ribavirin alone, provided a significant survival benefit to the influenza A virus-infected mice. The combination treatment protected the mice against severe influenza infection by attenuating the severe lung injury. The combined treatment also reduced the viral titers in mouse lungs and lung index, downregulated their immunocytokine levels, including IL-1ß and IL-18, and down regulated the NLRP3, especially the transcription and translation of caspase-1. Meanwhile NS group had significantly higher reactive oxygen species (ROS) expression which could was dramatically reduced by the treatment of RDN plus ribavirin. CONCLUSION: Our study showed that RDN combined with ribavirin could protect the mice, and reduce the lung immunopathologic damage caused by severe influenza pneumonia. The mechanism could be that it reduced ROS produce and inhibited NLRP3 inflammasome activation so that mainly lower the downstream inflammatory cytokines IL-1ß and IL-18.

Antiviral potential of garlic (Allium sativum) and its organosulfur compounds: A systematic update of pre-clinical and clinical data.

BACKGROUND: Garlic (Allium sativum L.) is a common herb consumed worldwide as functional food and traditional remedy for the prevention of infectious diseases since ancient time. Garlic and its active organosulfur compounds (OSCs) have been reported to alleviate a number of viral infections in pre-clinical and clinical investigations. However, so far no systematic review on its antiviral effects and the underlying molecular mechanisms exists. SCOPE AND APPROACH: The aim of this review is to systematically summarize pre-clinical and clinical investigations on antiviral effects of garlic and its OSCs as well as to further analyse recent findings on the mechanisms that underpin these antiviral actions. PubMed, Cochrane library, Google Scholar and Science Direct databases were searched and articles up to June 2020 were included in this review. KEY FINDINGS AND CONCLUSIONS: Pre-clinical data demonstrated that garlic and its OSCs have potential antiviral activity against different human, animal and plant pathogenic viruses through blocking viral entry into host cells, inhibiting viral RNA polymerase, reverse transcriptase, DNA synthesis and immediate-early gene 1(IEG1) transcription, as well as through downregulating the extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway. The alleviation of viral infection was also shown to link with immunomodulatory effects of garlic and its OSCs. Clinical studies further demonstrated a prophylactic effect of garlic in the prevention of widespread viral infections in humans through enhancing the immune response. This review highlights that garlic possesses significant antiviral activity and can be used prophylactically in the prevention of viral infections.

Identification of a Pharmacological Biomarker for the Bioassay-Based Quality Control of a Thirteen-Component TCM Formula (Lianhua Qingwen) Used in Treating Influenza A Virus (H1N1) Infection.

As chemical analysis for quality control (QC) of traditional Chinese medicine (TCM) formula is difficult to guarantee the effectiveness, a bioassay method that combines QC with evaluation of therapeutic effects has been developed to assess the TCM quality. Here, we chose a thirteen-component TCM formula, Lianhua Qingwen capsule (LHQW), as a representative sample, to explore the pivotal biomarkers for a bioassay and to investigate close association between QC and pharmacological actions. Initially, our results showed that chemical fingerprinting could not effectively distinguish batches of LHQW. Pharmacological experiments indicated that LHQW could treat influenza A virus (H1N1) infection in the H1N1 mouse model, as claimed in clinical trials, by improving pathologic alterations and bodyweight loss, and decreasing virus replication, lung lesions and inflammation. Furthermore, by using serum metabolomics analysis, we identified two important metabolites, prostaglandin F2α and arachidonic acid, and their metabolic pathway, arachidonic acid metabolism, as vital indicators of LHQW in treatment of influenza. Subsequently, macrophages transcriptomics highlighted the prominent role of cyclooxygenase-2 (COX-2) as the major rate-limiting enzyme in the arachidonic acid metabolism pathway. Finally, COX-2 was validated by in vivo gene expression and in vitro enzymatic activity with 43 batches of LHQW as a viable pharmacological biomarker for the establishment of bioassay-based QC. Our study provides systematic methodology in the pharmacological biomarker exploration for establishing the bioassay-based QC of LHQW or other TCM formulas relating to their pharmacological activities and mechanism.

[The Effects and Mechanisms of Dihydroartemisinin on Influenza A Virus H1N1 Induces TNF-α and IL-6 Expression in Bronchial Epithelial Cells].

OBJECTIVE: To investigate the effects of dihydroartemis (DHA) on influenza A virus (IAV) A/PR/8/34 (H1N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase (ERK) signaling pathway expression in bronchial epithelial cells. METHODS: The BEAS-2B cells were treated with different concentrations of DHA (i.e.,0， 12.5, 25，50 and 100 µmol/L) for 24 h and the effect of DHA on the viability of BEAS-2B cells were measure by CCK8 method. The BEAS-2B cells were absorbed with IAV for 1 h, and then were treated with different concentrations of DHA (i.e., 12.5, 25 and 50 µmol/L) for 24 h, meanwhile, the normal control group and IAV group were established. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were measured by real time quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), the expression levels of phospho-ERK (p-ERK) proteins were tested by Western blot (WB). Then, an ERK agonist (20 ng/mL) was used to treat BEAS-2B cells (the groups were divided into normal control group, DHA group, DHA+IAV group, ERK agonist group and DHA+IAV+ERK agonist group) for 24 h, and to observe the effect of DHA on inhibiting IAV induce the TNF-α, IL-6 and p-ERK expression in the BEAS-2B cells. RESULTS: The BEAS-2B cells viability was not significantly different from that of the normal control group after treatment with DHA (i.e., 12.5, 25, and 50 µmol/L). The expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein in IAV group were significantly up-regulated compared with that in the normal control group ( P<0.05), meanwhile, compared with the IAV group, the expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein showed dose-dependent decrease in IAV+DHA group ( P<0.05). However, ERK agonists attenuated the DHA inhibit IAV induced the proinflammatory factors TNF-α, IL-6 secretion and the p-ERK protein expression of ERK signaling pathway in BEAS-2B cells. CONCLUSION: These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2B cells through ERK signaling pathway.

In silico analysis and molecular characterization of Influenza A (H1N1) pdm09 virus circulating and causing major outbreaks in central India, 2009-2019.

BACKGROUND AND OBJECTIVES: Influenza A/H1N1pdm09 causes respiratory illness and remains a concern for public health. Since its first emergence in 2009, the virus has been continuously circulating in the form of its genetic variants. Influenza A/H1N1pdm09 surveillance is essential for uncovering emerging variants of epidemiologic and vaccine efficacy. The present study attempts in silico analysis and molecular characterization of Influenza A (H1N1) pdm09 virus circulating and causing major outbreaks in central India during 2009-2019. MATERIALS AND METHODS: We have investigated the antigenic drift analysis of 96 isolates' hemagglutinin (HA) gene sequences (59 central Indian and 37 local Indian and 28 global reference HA gene sequences) of Influenza A/H1N1pdm09 viruses from 2009 to 2019. The study includes mutational (Multiple sequence Alignment), phylogenetic (Maximum Likelihood Method), and statistical analysis (Covariance and correlation) of HA sequences submitted in NCBI, IRD and GISAID from central India. RESULTS: Phylogenetic analysis indicated maximum clustering of central Indian HA gene sequences in genogroup 6B. Analysis of amino acid sequence alignment revealed changes in receptor binding site (RBS). The frequency of S220T amino acid substitution was found to be high followed by S202T, K300E A273T, K180Q. The Karl Pearson correlation coefficient (r) and covariance between the number of mutations and the death toll was found +0.246 and +100.3 respectively. CONCLUSION: The study identifies the continuous genetic variations in the HA gene sequences of circulating Influenza A/H1N1pdm09 in central India from the year 2009 to 2019. Further suggesting importance of monitoring the gradual evolution of the virus with regards to an increase in virulence, pathogenicity and vaccine efficacy timely.

H1N1: perfil clínico-epidemiológico de pacientes atendidos em um hospital / H1N1: clinical and epidemiological profile of patients treated in a hospital

Objetivo: Avaliar casos de suspeita de gripe H1N1, bem como comparar aspectos epidemiológicos e clínicos dos pacientes com gripe H1N1 confirmada em relação àqueles não confirmados; analisar os critérios de gravidade clínica com relação à confirmação (ou não) da gripe H1N1 e seu desfecho (mortalidade); e criar um banco de dados para fins de comparação com a literatura nacional e mundial. Métodos: Estudo retrospectivo de coorte transversal realizado no período sazonal (outono e inverno) no ano de 2016. Foram analisados os prontuários, acessíveis e completos, de pacientes com suspeita clínica de H1N1, além daqueles com resultados definidos na sorologia. A partir dos dados coletados, foi elaborada tabela de análise epidemiológica, com informações clínicas, laboratoriais e sorológicas. Resultados: Destacam-se a média das faixas etárias mais acometidas de 48 anos, além dos sintomas mais comuns que foram dispneia, tosse e mialgia; as comorbidades foram hipertensão arterial sistêmica, cardiopatias, diabetes e doença pulmonar obstrutiva crônica. Conclusão: Este trabalho contribuiu com a caracterização do perfil epidemiológico regional e auxiliou na definição de indicadores de diagnóstico e gravidade, além de agregar à literatura conteúdos de caráter relevante. Este estudo está registrado como CAAE 58664016.2.0000.5515 na Plataforma Brasil. (AU)

Objective: To evaluate cases of suspected H1N1 flu, as well as to compare epidemiological and clinical aspects of patients with confirmed H1N1 influenza to those who were not confirmed; to analyze the criteria of clinical severity regarding the confirmation (or not) of H1N1 influenza, and its outcome (mortality); and to create a database to be compared with the national and world literature. Methods: This is a cross-sectional retrospective cohort study, carried out in the seasonal period ( fall/winter) of 2016. Accessible and complete medical records of patients with clinical suspicion of H1N1 were analyzed along with those with defined serology results. Based on the collected data, a table of epidemiological analysis was elaborated with clinical, laboratory and serological information. Results: The mean age of the most affected age groups was 48 years; the most common symptoms were dyspnea, cough and myalgia; and the comorbidities were systemic arterial hypertension, cardiopathies, diabetes, and chronic obstructive pulmonary disease. Conclusion: This work contributed to the characterization of the regional epidemiological profile, and helped in the definition of indicators of diagnosis and severity, besides adding relevant content to the literature. This study is registered as CAAE 58664016.2.0000.5515 at Plataforma Brasil. (AU)