scholarly journals Cellular and biochemical responses induced by Biotherapics prepared from intact influenza A (H3N2) and inactivated influenza A (H3N2) virus at 12x and 30x in the MDCK cells

2021 ◽  
Vol 10 (36) ◽  
pp. 170-171
Author(s):  
Camila Siqueira ◽  
Rafaela Mendonça ◽  
Venicio Veiga ◽  
Mariah Marcondes ◽  
Jose Nelson Couceiro ◽  
...  
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
H3n2 Virus ◽  
Biochemical Alterations ◽  
Virus Particles ◽  
Mtt Method ◽  
Transmission Electron ◽  
Viral Particles ◽  
The One ◽  
Homeopathic Remedies

Biotherapics are homeopathic remedies prepared from organic products that are chemically undefined and can be used for treatment of diseases like influenza. There are several classes of biotherapics and, among these, there are some called "living biotherapics" or "Roberto Costa’s Biotherapics". This study aimed to compare the cellular and biochemical effects of biotherapics prepared from intact influenza virus diluted in water and the one obtained from the same viral sample inactivated by ethanol 70% (v / v), both in the potencies of 12x and 30x. Transmission electron microscopy (TEM) analyses were performed on both preparations to assess the integrity of viral particles, which showed that ethanol 70% (v/v) induced a complete denaturation of viral particles. In contrast, the integrity of virus particles was preserved when water was used as the biotherapic solvent. Cellular and biochemical alterations induced by the preparations on MDCK cells were analyzed and compared with those induced by respective controls (water 30x-treated and untreated cells). Cellular viability analyzed by MTT method showed statistically significant differences (p

Rapid diagnosis of influenza A infection by direct immunofluorescence of nasopharyngeal aspirates in adults

1979 ◽  
Vol 9 (6) ◽  
pp. 688-692
Author(s):  
J A Daisy ◽  
F S Lief ◽  
H M Friedman
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Isolation ◽  
Fluorescent Antibody ◽  
Positive Culture ◽  
H3n2 Virus ◽  
Direct Immunofluorescence ◽  
The One ◽  
Immunofluorescent Test ◽  
Culture Negative

The efficacy for direct immunofluorescence of a commercial conjugate for influenza A virus prepared against whole A/Udorn (H3NS) virus was studied. The conjugate was specific for influenza A virus, but its sensitivity varied depending upon the strain of influenza A tested. Nasopharyngeal aspirates collected from 25 patients during an outbreak of influenza were examined for viral antigen with the conjugates and inoculated onto monkey kidney (MK) cells for virus isolation. Fifteen patients had isolates for influenza A/USSR/90/77 (H1N1); nasopharyngeal secretions were fluorescent antibody positive in 12. Fluorescent antibody was copositive with culture in 11/15 patients (73.3%) and conegative in 9/10 (90%). The one fluorescent antibody-positive, culture-negative patient had negative serology for influenza A and the fluorescent antibody result was considered to be a false positive. At a 1:10 dilution, the conjugate stained nasopharyngeal and MK cells infected with A/USSR (H1N1) 2 to 3+, whereas cells infected with H3N2 virus stained 4+. A conjugate made specifically against the ribonucleoprotein antigen, which is universal to all influenza A strains, may improve the sensitivity of the direct immunofluorescent test.

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 Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus

2008 ◽  
Vol 82 (23) ◽  
pp. 11869-11879 ◽  
Author(s):  
Edward C. Hutchinson ◽  
Martin D. Curran ◽  
Eliot K. Read ◽  
Julia R. Gog ◽  
Paul Digard
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genome Packaging ◽  
Virus Growth ◽  
Virus Particles ◽  
Synonymous Mutations ◽  
Coding Regions

ABSTRACT The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.

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 Influenza Virus Aerosols in the Air and Their Infectiousness

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Nikolai Nikitin ◽  
Ekaterina Petrova ◽  
Ekaterina Trifonova ◽  
Olga Karpova
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Hospital Admissions ◽  
Influenza Infection ◽  
Human Infection ◽  
Virus Concentration ◽  
Virus Particles ◽  
Viral Particles ◽  
Transmission And Distribution ◽  
The Relationship

Influenza is one of the most contagious and rapidly spreading infectious diseases and an important global cause of hospital admissions and mortality. There are some amounts of the virus in the air constantly. These amounts is generally not enough to cause disease in people, due to infection prevention by healthy immune systems. However, at a higher concentration of the airborne virus, the risk of human infection increases dramatically. Early detection of the threshold virus concentration is essential for prevention of the spread of influenza infection. This review discusses different approaches for measuring the amount of influenza A virus particles in the air and assessing their infectiousness. Here we also discuss the data describing the relationship between the influenza virus subtypes and virus air transmission, and distribution of viral particles in aerosol drops of different sizes.

scholarly journals Tyrosines in the Influenza A Virus M2 Protein Cytoplasmic Tail Are Critical for Production of Infectious Virus Particles

2010 ◽  
Vol 84 (17) ◽  
pp. 8765-8776 ◽  
Author(s):  
Michael L. Grantham ◽  
Shaun M. Stewart ◽  
Erin N. Lalime ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Alanine Scanning Mutagenesis ◽  
Particle Assembly ◽  
Virus Particles

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.

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 Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

2009 ◽  
Vol 83 (17) ◽  
pp. 8655-8661 ◽  
Author(s):  
Michael L. Grantham ◽  
Wai-Hong Wu ◽  
Erin N. Lalime ◽  
Maria E. Lorenzo ◽  
Sabra L. Klein ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Cysteine Residue ◽  
M2 Protein ◽  
Recombinant Viruses ◽  
Virus Particles

ABSTRACT The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

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