Drug resistance analysis of influenza virus to neuraminidase inhibitor from 2018 to 2019 in Hebei Province / 中华疾病控制杂志

Objective To analyze the resistance of influenza virus to neuraminidase inhibitors (NAI) in Hebei province during 2018-2019. Methods Virus were collected from the Hebei Influenza Surveillance Network during 2018-2019. A total of 36 confirmed influenza viruses (with 25 H1pdm09 and 11 H3N2) were selected to test resistance to oseltamivir and zanamivi with fluorescence (FL). Results All 36 influenza viruses tested were sensitive to oseltamivir and zanamivir. The median half maximal inhibitory concentration (IC50) for oseltamivir of H1pdm09 and H3N2 were of 0.50 nM (range 0.07-1.14 nM) and 0.25 nM (range 0.09-0.69 nM) respectively, while 0.29 nM (range 0.09-0.85 nM) and 0.87(range 0.17-1.81 nM) for zanamivir, all were within 10 fold IC50 of the reference virus (corresponding type). Conclusion All the tested influenza strains isolated in Hebei province during 2018-2019 were sensitive to NAI.

Neuraminidase Inhibitors from the Fruiting Body of Glaziella splendens

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1–5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2–5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 µM for 3 and 46.5, 50.4, and 29.9 µM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.

Resistance of influenza virus in Henan Province to neuraminidase inhibitor from 2017 to 2018 / 中华疾病控制杂志

Objective To analyze the resistance of influenza virus to neuraminidase inhibitors (NAI) in Henan province during 2017-2018. Methods Virus were collected from the Henan Influenza Surveillance Network during 2017-2018. 36 confirmed influenza virus(with 15 H1pdm09，6 H3N2 and 15 B) were selected to test resistance to oseltamivir and zanamivi with fluorescence(FL). The NAI sensitive reference viruses were A/California/12/2012(H1pdm09)-275H,A/Beijing Haidian/1942/2014(H3N2)-119E and B/Rochester/02/2001-198D. The NAI resistant reference viruses were A/ Texas/23/2012(H1pdm9)-H275Y, A/Texas/12/2007(H3N2)-E119V and B/Rochester/02/2001-D198N. Results The half maximal inhibitory concentration(IC50) of A/California/12/2012(H1pdm09)-275H, A/Beijing-Haidian/1942/2014(H3N2)-119E and B/Rochester/02/2001-198D for oseltamivir were 0.29 nmol/L (nM),0.10 nM and 12.71 nM, and for zanamivir were 0.2 nM, 0.49 nM and 0.33 nM respectively. The IC50 for oxastatin of H1pdm09 and H3N2 ranged from (0.28-1.37 nM) and (0.08-0.17 nM) respectively, the IC50 for zanamivir ranged from (0.15-0.49 nM) and (0.12-0.22 nM), all was within 10 fold IC50 of the reference virus(corresponding type); the IC50 value of type B for oseltamivir and zanamivir ranged from (11.83-24.59 nM) and (0.48-1.25 nM), all was within 5 fold IC50 of the reference virus. Conclusion All the tested influenza strains isolated in Henan province during 2017-2018 were sensitive to NAI.

Clinical findings of 16 cases of neonatal influenza / 临床儿科杂志

Objective To investigate the clinical features, laboratory diagnostics, treatments, and prognosis of neonates infected with influenza. Methods The clinical data of 16 neonates diagnosed as influenza admitted to the neonatal ward from January 2015 to May 2017 were retrospectively analyzed. Results Of the 16 neonates, 11 were male and 5 were female. Mean age was 18.5 days. 75% (12/16) of them were reported to be exposed to family members with common cold- like symptoms before hospitalization. Clinical manifestations included nasal obstruction (11/16), fever (10/16), cough (10/16), and rhinorrhea (8/16). Influenza antigen rapid detection (colloidal gold method) was positive in all cases. Influenza immunofluorescence assays were performed in 15 cases, only 6.67% (1/15) was positive. Sputum culture was performed in 13 cases, 8 of which were positive. Of them, 75% (12/16) neonates were diagnosed with pneumonia. Only 12.5% (2/16) neonates were treated with neuraminidase inhibitor. All cases recovered well and were discharged after antibiotic treatment. Conclusions Neonates contacted with family members displaying common cold-like symptoms should be examined for influenza in time. The common clinical manifestations include catarrhal symptoms, fever and cough. The sensitivity of the influenza immunofluorescence assay is lower as compared with the colloidal gold method. Pneumonia may often be developed in neonatal influenza. The prognosis of neonatal influenza is satisfactory if treated.

Neuraminidase Inhibitors from the Fruiting Body of Phellinus igniarius

During our ongoing investigation of neuraminidase inhibitors from medicinal fungi, we found that the fruiting bodies of Phellinus igniarius exhibited significant inhibitory activity against neuraminidase from recombinant H3N2 influenza viruses. Two active compounds were isolated from the methanolic extract of P. igniarius through solvent partitioning and Sephadex LH-20 column chromatography. The active compounds were identified as phelligridins E and G on proton nuclear magnetic resonance (¹H NMR) and electrospray ionization mass measurements. These compounds inhibited neuraminidases from recombinant rvH1N1, H3N2, and H5N1 influenza viruses, with IC₅₀ values in the range of 0.7~8.1 µM.

Susceptibility of influenza B viruses to neuraminidase inhibitors during 2014 to 2015 in mainland China / 中华微生物学和免疫学杂志

Objective To analyze the phenotypic characteristics of antiviral-resistant influenza B viruses circulating in mainland China and to analyze the susceptibility of influenza B viruses to neuraminidase inhibitors ( NAIs) . Methods Antiviral-resistant phenotyping test was performed to analyze the NAI suscep-tibility of 1 386 influenza B viruses isolated in mainland China from April 2014 to March 2015, including the test of susceptibility to oseltamivir and zanamivir. Results All of the 94 B-Victoria lineage viruses were sensitive to oseltamivir and zanamivir. Of all 1 292 B-Yamagata lineage viruses tested, 1 virus showed re-duced sensitivity to oseltamivir with NA gene containing I221T amino acid mutation, 10 viruses showed re-duced sensitivity to zanamivir with 4 having D197N amino acid mutation in NA gene, 3 viruses showed re-duced sensitivity to both oseltamivir and zanamivir with NA gene possessing D197N amino acid mutation and 1 virus carrying the A245T amino acid mutation in NA gene showed reduced sensitivity to oseltamivir and highly reduced sensitivity to zanamivir. Conclusion The majority of influenza B viruses circulating in main-land China during 2014 to 2015 were sensitive to NAIs, which indicated that NAIs could be used continually for clinical treatment of patients with influenza. Sustained monitoring of antiviral susceptibility of influenza B viruses should be emphasized for timely detection of antiviral resistant viruses and more attention should be paid to the D197N mutations in NA gene of influenza B viruses.

Advances in anti-influenza virus of neuraminidase inhibitors / 中国药学杂志

Neuraminidase (NA) is a mushroom-shaped and tetramer structural envelope glycoprotein on the surface of the influenza virus. NA inhibitors can inhibit highly pathogenic influenza virus subtypes and have good safety and drug resistance, hence they are widely used for the prevention and treatment of influenza virus. Based on the present domestic and foreign literatures in this field, this paper summarizes the research status of neuraminidase inhibitors classification and structure-activity relationships. It will help us make better use of existing conditions to design and synthesize better active and more selective anti-influenza drugs.

Antiviral therapy for severe viral pneumonia / 中国小儿急救医学

Adenovirus,respiratory syncytial virus,influenza virus type A and B,cytomegalovirus and EB virus are the mainly etiology of severe pneumonia in children.New type of virus,such as influenza-H1N1 virus,avian influenza virus(H5N1 or H7N9) can also be epidemic in pediatric population.Ribavirin is effective drugs in the treatment of respiratory syncytial virus and adenovirus pneumonia.Acyclovir or ganciclovir is used for EB virus or immune deficiency and irnmunosuppressive patients with CMV pneumonia.Current opinin strongly recommend treatment with oral oseltamivir as soon as possible in influenza and seasonal influenza.Oseltamivir reduces the severity,duration of the symptoms of influenza,and reduces the frequency of secondary illnesses and exacerbation of underlying conditions.Zanamivir and peramivir may be effective in patients infected with influenza virus,including oseltamivir-resistant virus.Some Chinese medicine such as maxingshigan-yinqiaosan can obtain similar effect of oseltamivir in treatment of influenza virus infection.

Neuraminidase Inhibitors from the Fermentation Broth of Phellinus linteus

During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 microM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections.

Neuraminidase Inhibitors from the Culture Broth of Phellinus linteus

During the search for neuraminidase inhibitors from medicinal fungi, we found that the culture broth of Phellinus linteus exhibited potent inhibitory activity. Solvent partition, Sephadex LH-20 column chromatography, and high-performance liquid chromatography (HPLC) were performed for purification of two active substances from the culture broth. According to 1H NMR measurements and comparison of HPLC retention times with those of authentic compounds, their chemical structures were identified as hispidin and hypholomine B. Compounds (hispidin) 1 and 2 (hypholomine B) inhibited neuraminidase, with IC50 values of 13.1 and 0.03 microM, respectively.

Surveillance for neuraminidase inhibitor resistance of seasonal H1N1 influenza A viruses isolated in Shenzhen during 2008 to 2009 / 中华微生物学和免疫学杂志

Objective To analyze neuraminidase(NA) inhibitor resistance of seasonal H1N1 influenza A viruses isolated in Shenzhen during 2008 to 2009. Methods The NA gene of these viruses were sequenced. Phylogenetic analysis of the sequences was performed with Mega3. 1 software. Results In 2008, most isolates of the seasonal H1 N1 virus were susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance had been detected in 92.6% of the strains isolated in 2009. Furthermore, a strain with Q136K was found, which showed the resistance to Zanamivir. Conclusion In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in influenza virus should be a priority.

Quantification of peramivir in rat plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization and its application in pharmacokinetics / 军事医学科学院院刊

Objective:To develop a liquid chromatography/tandem mass spectrometry employing precolumn derivatization method for determination of peramivir in rat plasma.Methods: The sample preparation consisted of a protein precipitation extraction followed by derivatization with hydrochloric acid (10 mol/L) methanol (10∶90, v/v) and determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The positive ion SRM mode was performed and the precursor-to-the-product ion transitions of m/z 343→284 and 299→152 were used to measure the derivative of peramivir and Ro 64-0802 (I.S.). The chromatographic separation was achieved using a Zorbax RX-C8 (2.1 mm × 150 mm × 5 μm) analytical column with an isocratic mobile phase composed of acetonitrile-water-formic acid (30∶70∶0.1, v/v/v, 0.2 ml/min). Results:The method was linear over a concentration range of 10 to 10 000 ng/ml. The LLOQ was 10 ng/ml. The average inter-day/intra-day precision values ranged from 5.0% to 7.1%, respectively, while the accuracy values ranged from 89.9% to 106.1%.Conclusion: In this method, retention time is greatly improved, the matrix effects are decreased by chemical derivatization. This method has been successfully applied to the preclinical and clinical research of peramivir.

Characterization of Hemagglutinin and Neuraminidase Genes and Oseltamivir Resistance of Influenza Viruses Isolated in Korea

To investigate the pattern of drug-resistance of human influenza virus (A/H1N1) isolated in Korea during 2001~2002, the sequence analysis of hemagglutinin (HA) and neuraminidase (NA) genes and cell-based assay against neuraminidase inhibitor (NI) were performed. Analyses on the nucleotide sequences of NA genes showed that Korean isolates had 98.2 to 98.5% homology with that of the vaccine strain in 2001~2002 season, A/New Caledonia/20/99-like strain. However, there were no significant amino acid substitutions related to the drug-resistance such as E119V, R152K, I222R/Q, H274Y, and R292K. In the sequences of HA gene, no differences were observed on the major antigenic sites as well as the motifs related to the drug resistance. 50% inhibitory concentration (IC50) value against oseltamivir, one of NA inhibitors widely used in the treatment for the influenza, was determined by WST-1 assay. The SI values of Korean isolates against oseltamivir were 7.2 to 383.3, showing that these isolates displayed relatively low SI value against the drug. This result provides the useful information for the surveillance of drug-resistant influenza virus and the control of influenza in Korea.