scholarly journals Cell Culture-Selected Substitutions in Influenza A(H3N2) Neuraminidase Affect Drug Susceptibility Assessment

2013 ◽  
Vol 57 (12) ◽  
pp. 6141-6146 ◽  
Author(s):  
Daisuke Tamura ◽  
Ha T. Nguyen ◽  
Katrina Sleeman ◽  
Marnie Levine ◽  
Vasiliy P. Mishin ◽  
...  
Keyword(s):  
Mdck Cell ◽  
Influenza A ◽  
Mdck Cells ◽  
Clinical Sample ◽  
Fold Increase ◽  
Drug Susceptibility ◽  
H3n2 Virus ◽  
Oseltamivir Resistance ◽  
Viral Propagation ◽  
Drug Resistance Profile

ABSTRACTAssessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n= 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.

Replication and transmission of an influenza A(H3N2) virus harboring the polymerase acidic I38T substitution, in guinea pigs.

2021 ◽  
Vol 102 (10) ◽  
Author(s):  
Zeineb Mhamdi ◽  
Julie Carbonneau ◽  
Marie-Christine Venable ◽  
Mariana Baz ◽  
Yacine Abed ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
A549 Cells ◽  
Viral Fitness ◽  
Viral Population ◽  
H3n2 Virus ◽  
The Impact

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml−1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

scholarly journals Surveillance for Neuraminidase Inhibitor Resistance among Human Influenza A and B Viruses Circulating Worldwide from 2004 to 2008

2008 ◽  
Vol 52 (9) ◽  
pp. 3284-3292 ◽  
Author(s):  
Tiffany G. Sheu ◽  
Varough M. Deyde ◽  
Margaret Okomo-Adhiambo ◽  
Rebecca J. Garten ◽  
Xiyan Xu ◽  
...  
Keyword(s):  
United States ◽  
Influenza A ◽  
Neuraminidase Inhibitor ◽  
The United States ◽  
H3n2 Virus ◽  
Influenza B ◽  
Close Monitoring ◽  
Oseltamivir Resistance ◽  
H3n2 Influenza ◽  
Inhibitor Resistance

ABSTRACT The surveillance of seasonal influenza virus susceptibility to neuraminidase (NA) inhibitors was conducted using an NA inhibition assay. The 50% inhibitory concentration values (IC50s) of 4,570 viruses collected globally from October 2004 to March 2008 were determined. Based on mean IC50s, A(H3N2) viruses (0.44 nM) were more sensitive to oseltamivir than A(H1N1) viruses (0.91 nM). The opposite trend was observed with zanamivir: 1.06 nM for A(H1N1) and 2.54 nM for A(H3N2). Influenza B viruses exhibited the least susceptibility to oseltamivir (3.42 nM) and to zanamivir (3.87 nM). To identify potentially resistant viruses (outliers), a threshold of a mean IC50 value + 3 standard deviations was defined for type/subtype and drug. Sequence analysis of outliers was performed to identify NA changes that might be associated with reduced susceptibility. Molecular markers of oseltamivir resistance were found in six A(H1N1) viruses (H274Y) and one A(H3N2) virus (E119V) collected between 2004 and 2007. Some outliers contained previously reported mutations (e.g., I222T in the B viruses), while other mutations [e.g., R371K and H274Y in B viruses and H274N in A(H3N2) viruses) were novel. The R371K B virus outlier exhibited high levels of resistance to both inhibitors (>100 nM). A substantial variance at residue D151 was observed among A(H3N2) zanamivir-resistant outliers. The clinical relevance of newly identified NA mutations is unknown. A rise in the incidence of oseltamivir resistance in A(H1N1) viruses carrying the H274Y mutation was detected in the United States and in other countries in the ongoing 2007 to 2008 season. As of March 2008, the frequency of resistance among A(H1N1) viruses in the United States was 8.6% (50/579 isolates). The recent increase in oseltamivir resistance among A(H1N1) viruses isolated from untreated patients raises public health concerns and necessitates close monitoring of resistance to NA inhibitors.

scholarly journals High antiviral effect of TiO2·PL–DNA nanocomposites targeted to conservative regions of (−)RNA and (+)RNA of influenza A virus in cell culture

2016 ◽  
Vol 7 ◽  
pp. 1166-1173 ◽  
Author(s):  
Asya S Levina ◽  
Marina N Repkova ◽  
Elena V Bessudnova ◽  
Ekaterina I Filippova ◽  
Natalia A Mazurkova ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Titanium Dioxide Nanoparticles ◽  
Antiviral Effect ◽  
H3n2 Virus ◽  
Dna Fragments ◽  
Efficient System ◽  
Dna Fragment ◽  
Viral Subtypes

Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL)-containing oligonucleotides. Results: The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−)RNA and (+)RNA of segment 5 of influenza A virus (IAV) were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−)RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+)RNA and the corresponding region of (−)RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−)RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+)RNA regions. Conclusion: The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−)RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+)RNA.

scholarly journals The whole-genome sequencing in predicting Mycobacterium tuberculosis drug susceptibility and resistance in Papua, Indonesia

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yustinus Maladan ◽  
Hana Krismawati ◽  
Tri Wahyuni ◽  
Ratna Tanjung ◽  
Kamla Awaludin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Mapping ◽  
Clinical Sample ◽  
Drug Susceptibility ◽  
Whole Genome ◽  
Tb Treatment ◽  
Drug Resistance Profile

Abstract Background Tuberculosis is one of the deadliest disease caused by Mycobacterium tuberculosis. Its treatment still becomes a burden for many countries including Indonesia. Drug resistance is one of the problems in TB treatment. However, a development in the molecular field through Whole-genome sequencing (WGS) can be used as a solution in detecting mutations associated with TB- drugs. This investigation intended to implement this data for supporting the scientific community in deeply understanding any TB epidemiology and evolution in Papua along with detecting any mutations in genes associated with TB-Drugs. Result A whole-genome sequencing was performed on the random samples from TB Referral Laboratory in Papua utilizing MiSeq 600 cycle Reagent Kit (V3). Furthermore, TBProfiler was used for genome analysis, RAST Server was employed for annotation, while Gview server was applied for BLAST genome mapping and a Microscope server was implemented for Regions of Genomic Plasticity (RGP). The largest genome of M. tuberculosis obtained was at the size of 4,396,040 bp with subsystems number at 309 and the number of coding sequences at 4326. One sample (TB751) contained one RGP. The drug resistance analysis revealed that several mutations associated with TB-drug resistance existed. In details, mutations of rpoB gene which were identified as S450L, D435Y, H445Y, L430P, and Q432K had caused the reduced effectiveness of rifampicin; while the mutases in katG (S315T), kasA (312S), inhA (I21V), and Rv1482c-fabG1 (C-15 T) genes had contributed to the resistance in isoniazid. In streptomycin, the resistance was triggered by the mutations in rpsL (K43R) and rrs (A514C, A514T) genes, and, in Amikacin, its resistance was led by mutations in rrs (A514C) gene. Additionally, in Ethambutol and Pyrazinamide, their reduced effectiveness was provoked by embB gene mutases (M306L, M306V, D1024N) and pncA (W119R). Conclusions The results from whole-genome sequencing of TB clinical sample in Papua, Indonesia could contribute to the surveillance of TB-drug resistance. In the drug resistance profile, there were 15 Multi Drugs Resistance (MDR) samples. However, Extensively Drug-resistant (XDR) samples have not been found, but samples were resistant to only Amikacin, a second-line drug.

Madin Darby canine kidney cell-lines used in influenza virus production has resulted in canine 28s being integrated in the influenza virus genome - evidence from Influenza A patients in Hong Kong

2020 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza Vaccine ◽  
Mdck Cell ◽  
Influenza A ◽  
Mdck Cells ◽  
Virus Genome ◽  
Kidney Cells ◽  
Darby Canine Kidney ◽  
Canine Kidney

In 1989, 54 nucleotides from chicken 18s were seen to be inserted into the haemagglutinin gene of an influenza virus increasing viral pathogenicity [1]. Previously, I have reported human 18s sequences (from sequence vectors) appended to the influenza virus genome in Covid19 patients from Wuhan and Hong Kong [2]. These human ribosomal sequences are supposed to increase the transcription of the virus in the human cell, and thus will be more pathogenic. Here, I report the circulation of Influenza A genomes with 28s from canine integrated, showing that the escape of lab-made viruses is quite prevalent.Canine 28s sequence appended to flu genomesA recent (2020,Accid:PRJNA605947) study from the University of Hong Kong that did Nanopore sequencing to find novel targets for detection and surveillance of Influenza A viruses [3] shows the integration of canine 28s (Fig 1) sequence to the flu genome. The full read (SRR11067307.3179,SI:fullread.fa) splits into the flu virus (1-1605, SI:flu.baltimore.fa,(Baltimore/R0197/2017(H1N1)) nucleocapsid protein (NP) gene) and canine 28 (1606-1973, SI:canine.28s.fa Accid:XR 004817748.1, Canis lupus dingo 28S)). There is no reason to find canine 28s in clinical samples, barring the fact that canine kidney cells are used to manufacture the virus for several applications, including vaccines.Madin Darby canine kidney cells (MDCK) - why MDCK?The advantages of using MDCK influenza production is well known [4]. MDCK is better in the replication of live attenuated influenza viruses than most other cell lines (like Vero), thus yielding more virus in large-scale production of influenza virus [5]. The virus replicates rapidly in MDCK to ‘produce high titers in MDCK cells in as few as 3 to 10 passages, i.e., in 10–30 days’ [4]. Also, MDCK cells are also good for the production of certain influenza B virus vaccines, and MDCK cell-derived components are not allergenic [6]. Vaccines made using MDCK cellsInfluvac, a split virus vaccine produced in adherent MDCK cells, in the Netherlands in 1999 [7]. The use of MDCK cells (MedImmune) for production of live attenuated influenza vaccine in both serum containing and serum-free media was found to be more efficient [8]. Another trivalent MDCK cell culture-derived influenza vaccine is Optaflu [9]. ”Flucelvax Quadrivalent is the only cell-based inactivated flu vaccine that has been licensed by the FDA for use in the United States.” (https://www.cdc.gov/flu/prevent/cell-based.htm)

scholarly journals An influenza reassortant with polymerase of pH1N1 and NS gene of H3N2 influenza A virus is attenuated in vivo

2012 ◽  
Vol 93 (5) ◽  
pp. 998-1006 ◽  
Author(s):  
Holly Shelton ◽  
Matt Smith ◽  
Lorian Hartgroves ◽  
Peter Stilwell ◽  
Kim Roberts ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Gene Segment ◽  
Influenza Viruses ◽  
Interferon Response ◽  
H3n2 Virus ◽  
M Gene ◽  
Ns Gene

Influenza viruses readily mutate by accumulating point mutations and also by reassortment in which they acquire whole gene segments from another virus in a co-infected host. The NS1 gene is a major virulence factor of influenza A virus. The effects of changes in NS1 sequence depend on the influenza polymerase constellation. Here, we investigated the consequences of a virus with the polymerase of pandemic H1N1 2009 acquiring an NS gene segment derived from a seasonal influenza A H3N2 virus, a combination that might arise during natural reassortment of viruses that currently circulate in humans. We generated recombinant influenza viruses with surface HA and NA genes and matrix M gene segment from A/PR/8/34 virus, but different combinations of polymerase and NS genes. Thus, any changes in phenotype were not due to differences in receptor use, entry, uncoating or virus release. In Madin–Darby canine kidney (MDCK) cells, the virus with the NS gene from the H3N2 parent showed enhanced replication, probably a result of increased control of the interferon response. However, in mice the same virus was attenuated in comparison with the virus containing homologous pH1N1 polymerase and NS genes. Levels of viral RNA during single-cycles of replication were lower for the virus with H3N2 NS, and this virus reached lower titres in the lungs of infected mice. Thus, virus with pH1N1 polymerase genes did not increase its virulence by acquiring the H3N2 NS gene segment, and MDCK cells were a poor predictor of the outcome of infection in vivo.

scholarly journals Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin

2010 ◽  
Vol 84 (9) ◽  
pp. 4277-4288 ◽  
Author(s):  
Evelien Vanderlinden ◽  
Fusun Göktaş ◽  
Zafer Cesur ◽  
Matheus Froeyen ◽  
Mark L. Reed ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Structure Activity Relationship ◽  
Activity Relationship ◽  
H3n2 Virus ◽  
Wild Type Virus ◽  
Erythrocyte Hemolysis ◽  
Structure Activity ◽  
Virus Fusion

ABSTRACT A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 μM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.

scholarly journals Biochemical responses induced by Biotherapics prepared from intact influenza A (H3N2) and inactivated influenza A (H3N2) virus at 12x and 30x in MDCK cells and RAW-264-7 macrophages

2021 ◽  
Vol 11 (40) ◽  
pp. 180-181
Author(s):  
Camila Siqueira ◽  
Rafaela De Mendonça ◽  
Venício Da Veiga ◽  
Mariah Marcondes ◽  
Juliana Grechi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mtt Assay ◽  
Influenza A ◽  
Cellular Response ◽  
Mdck Cells ◽  
Experimental Situation ◽  
Neutral Red ◽  
H3n2 Virus ◽  
Mitochondrial Activity ◽  
Starting Point

Background: "Roberto Costa’s Biotherapics" are homeopathic remedies prepared from intact microorganisms which have been proposed for treatment of diseases like influenza. Aim: This study aimed to compare the biochemical effects, in MDCK cells and RAW-264-7 macrophages, of biotherapics prepared from intact influenza virus diluted in water as well as from a sample of the same virus inactivated by ethanol 70% (v / v), both in the homeopathic potencies of 12x and 30x. Water 30x, non-dynamized water and cells without treatment (control cells) were used as control. Methodology: Treatments were performed by incubating MDCK cells with DMEM medium added in a 1:10 ratio for 6 times (3 different aliquots per day) or 18 times (up to 4 aliquots per day) in each experimental situation. Each aliquot was added with an interval of at least 2 hours. After that, the mitochondrial activity of MDCK cells was analyzed by MTT assay. The effects of treatments with intact biotherapics on MDCK cells respiratory parameters were studied using high resolution respirometry (Oroboros Oxygraph-O2K). RAW-264-7 macrophages were treated with intact and inactivated biotherapic 30x (4 treatments, 24 hours) to verify the nitric oxide production. These macrophages were also submitted to MTT assay. Results: Both biotherapic preparations 1x (intact and inactivated virus sample) were analyzed by transmission electronic microscopy. The presence of virus particles was detected when water was used as solvent. The use of ethanol as biotherapic solvent induced complete virus lysis. There was no alteration in cell osmolarity revealed by neutral red assay, when 10% of each test solution was used. Cellular viability analyzed by MTT method increased when MDCK cells were treated with 18 stimuli of inactivated biotherapic 30x when compared to intact biotherapic 30x (p0.05) were detected when these cells were compared to control cells. The maximum respiratory capacity of MDCK cells increased after treatment with 18 stimuli of intact biotherapic 30x when compared to control cells. However, no statistically significant differences (p>0.05) induced by biotherapics in macrophage cells were observed by MTT and nitric oxide assays. Moreover, a reduction in nitric oxide was observed in macrophages treated with dynamized water when compared to control cells. Conclusions: These results indicate that the method of biotherapic compounding (intact or inactivated virus as starting point) can modify the cellular parameters with the tendency to increase cellular response with longer treatments and higher potencies.

scholarly journals Generation of a Reassortant Influenza A Subtype H3N2 Virus Expressing Gaussia Luciferase

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 665 ◽  
Author(s):  
Lin Wang ◽  
Qinghua Cui ◽  
Xiujuan Zhao ◽  
Ping Li ◽  
Yanyan Wang ◽  
...  
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
H3n2 Virus ◽  
Influenza A Viruses ◽  
Madin Darby Canine Kidney ◽  
Gaussia Luciferase ◽  
Ns Gene ◽  
Darby Canine Kidney ◽  
Canine Kidney

Reporter influenza A viruses (IAVs) carrying fluorescent or luminescent genes provide a powerful tool for both basic and translational research. Most reporter IAVs are based on the backbone of either subtype H1N1 viruses, A/Puerto Rico/8/1934 (PR8) or A/WSN/1933, but no reporter subtype H3N2 virus is currently available to our knowledge. Since the IAV subtype H3N2 co-circulates with H1N1 among humans causing annual epidemics, a reporter influenza A subtype H3N2 virus would be highly valuable. In this study, the segments of A/Wyoming/3/03 (NY, H3N2) virus encoding hemagglutinin and neuraminidase, respectively, were reassorted with the six internal genes of PR8 where the NS gene was fused with a Gaussia luciferase (Gluc) gene. Using reverse genetics, NY-r19-Gluc, a replication competent reassortant influenza A subtype H3N2 virus expressing reporter Gluc was successfully generated. This reporter virus is stable during replication in Madin-Darby canine kidney (MDCK) cells, and preliminary studies demonstrated it as a useful tool to evaluate antivirals. In addition, NY-r19-Gluc virus will be a powerful tool in other studies including the application of diagnostic and therapeutic antibodies as well as the evaluation of novel vaccines.

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