scholarly journals Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 794
Author(s):  
Beatriz Vidaña ◽  
Pamela Martínez-Orellana ◽  
Jaime Martorell ◽  
Massimiliano Baratelli ◽  
Jorge Martínez ◽  
...  
Keyword(s):  
Influenza A ◽  
Clinical Signs ◽  
Viral Fitness ◽  
Type I ◽  
Lung Pathology ◽  
Wild Type ◽  
Viral Kinetics ◽  
Influenza A H1n1

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.

scholarly journals Evaluation of Recombinant 2009 Pandemic Influenza A (H1N1) Viruses Harboring Zanamivir Resistance Mutations in Mice and Ferrets

2013 ◽  
Vol 57 (4) ◽  
pp. 1784-1789 ◽  
Author(s):  
Andrés Pizzorno ◽  
Yacine Abed ◽  
Chantal Rhéaume ◽  
Xavier Bouhy ◽  
Guy Boivin
Keyword(s):  
Influenza A ◽  
Fold Increase ◽  
Viral Fitness ◽  
Wild Type ◽  
Resistance Mutations ◽  
Nasal Wash ◽  
Influenza A H1n1 ◽  
Ratio Versus ◽  
Viral Titers

ABSTRACTRecombinant influenza A(H1N1)pdm09 wild-type (WT) and zanamivir-resistant E119G and Q136K neuraminidase mutants were generated to determine their enzymatic and replicative propertiesin vitro, as well as their infectivity and transmissibility in mice and ferrets. Viral titers of recombinant E119G and Q136K mutants were significantly lower than those of the WT in the first 36 h postinoculation (p.i.)in vitro. The E119G and Q136K mutations were both associated with a significant reduction of total neuraminidase (NA) activity at the cell surface of 293T cells, with relative total NA activities of 14% (P< 0.01) and 20% (P< 0.01), respectively, compared to the WT. The E119G mutation significantly reduced the affinity (8-fold increase inKm) but not theVmax. The Q136K mutation increased the affinity (5-fold decrease inKm) with a reduction inVmax(8%Vmaxratio versus the WT). In mice, infection with the E119G and Q136K mutants resulted in lung viral titers that were significantly lower than those of the WT on days 3 p.i. (3.4 × 106± 0.8 × 106and 2.1 × 107± 0.4 × 107PFU/ml, respectively, versus 8.8 × 107± 1.1 × 107;P< 0.05) and 6 p.i. (3.0 × 105± 0.5 × 105and 8.6 × 105± 1.4 ×105PFU/ml, respectively, versus 5.8 × 107± 0.3 × 107;P< 0.01). In experimentally infected ferrets, the E119G mutation rapidly reverted to the WT in donor and contact animals. The Q136K mutation was maintained in ferrets, although nasal wash viral titers from the Q136K contact group were significantly lower than those of the WT on days 3 to 5 p.i. Our results demonstrate that zanamivir-resistant E119G and Q136K mutations compromise viral fitness and transmissibility in A(H1N1)pdm09 viruses.

scholarly journals In Vitro and In Vivo Characterization of Novel Neuraminidase Substitutions in Influenza A(H1N1)pdm09 Virus Identified Using Laninamivir-Mediated In Vitro Selection

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Khristine Kaith S. Lloren ◽  
Jin Jung Kwon ◽  
Won-Suk Choi ◽  
Ju Hwan Jeong ◽  
Su Jeong Ahn ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Deep Sequencing ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Viral Fitness ◽  
Pdm09 Virus ◽  
Influenza A H1n1 ◽  
Na Gene

ABSTRACT Neuraminidase (NA) inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs—particularly newly introduced treatments—are critical to the management of viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (i.e., D199E and P458T) were shown to exhibit resistance to more than one NAI. Of note, mutants possessing P458T—which is located outside of the catalytic or framework residue of the NA active site—exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower than that of the wild type (WT). Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with the WT. Reverse mutations to the WT were observed in lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified could possibly emerge in influenza A(H1N1)pdm09 viruses during laninamivir therapy and the viruses could have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading. IMPORTANCE With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAI in several countries; few data exist related to the in vitro selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis of the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which conferred resistance to NAIs and which had an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.

scholarly journals Innate triggering and antiviral effector functions of activin A

2021 ◽  
Author(s):  
Kinda Al-Hourani ◽  
Narayan Ramamurthy ◽  
Emanuele Marchi ◽  
Ruth M Eichinger ◽  
Lian N Lee ◽  
...  
Keyword(s):  
Viral Infection ◽  
Influenza A ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Activin A ◽  
Type I ◽  
Type I Ifn ◽  
Effector Functions

First-line defence against viral infection is contingent upon rapid detection of conserved viral structural and genomic motifs by germline-encoded pattern recognition receptors, followed by activation of the type I IFN system and establishment of an intracellular antiviral state. Novel antiviral functions of bone morphogenetic protein and related activin cytokines, acting in conjunction with, and independently of, type I IFN, have recently been described. Activin A mediates multiple innate and adaptive immune functions, including antiviral effects. However, how such effects are mediated and how activin might be triggered by viral infection have not been defined. Here we addressed this in vivo and in vitro, in humans and mice. Transcriptomic analyses delineated strikingly congruent patterns of gene regulation in hepatocytes stimulated with recombinant activin A and IFNα in vitro. Activin A mRNA, encoded by INHBA, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and in human peripheral blood mononuclear cells. In vivo, infection of mice with influenza A also upregulated Inhba mRNA in the lung; this local upregulation of Inhba is retained in MAVS knockout mice, indicating a role for non-RIG-I-like receptors in its induction. Activin induction and signalling were also detectable in patients with chronic viral hepatitis. Together, these data suggest Activin A is triggered in parallel with type I IFN responses and can trigger related antiviral effector functions. This model has implications for the development of targeted antiviral therapies, in addition to revealing novel facets of activin biology.

scholarly journals Optimization of the Production Technology of Oxidized Cyclodextrin Bisulfite

Processes ◽  
2019 ◽  
Vol 7 (7) ◽  
pp. 426
Author(s):  
Yana-Ya Kostyro ◽  
Anastasiya Soldatenko ◽  
Alexey Levchuk
Keyword(s):  
Influenza A ◽  
Cross Flow ◽  
Active Pharmaceutical Ingredient ◽  
Environmental Friendliness ◽  
Whole Process ◽  
In Vivo Experiments ◽  
Cross Flow Filtration ◽  
Influenza A H1n1

The A.E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences has developed an original active pharmaceutical ingredient based on an oxidized cyclodextrin oligosaccharide, which is a bisulfite derivative. Conducted pharmacological studies proved its antiviral activity in vitro and in vivo experiments against the influenza A (H1N1) virus. The aim of this work was to optimize the technology of obtaining the active pharmaceutical ingredient based on the bisulfite derivative of oxidized cyclodextrin to increase the efficiency and safety of the process. For this, a scaled method of oligosaccharide oxidation was tested on pilot plants in accordance with the requirements of green chemistry. As a result, the reaction time was reduced from three to five days (laboratory conditions) to 1.5 h, and the safety and environmental friendliness of process was ensured. The use of cross-flow filtration and the method of freeze-drying eliminated 96% of ethyl alcohol, reduced the laboriousness and energy consumption of the technological operations for purification and isolation of the final product, and also increased the productivity of the whole process (output increased to 98%). The results are confirmed by data obtained by physicochemical methods.

scholarly journals Heterosubtypic Protection Conferred by the Human Monoclonal Antibody PN-SIA28 against Influenza A Virus Lethal Infections in Mice

2015 ◽  
Vol 59 (5) ◽  
pp. 2647-2653 ◽  
Author(s):  
Miguel Retamal ◽  
Yacine Abed ◽  
Chantal Rhéaume ◽  
Francesca Cappelletti ◽  
Nicola Clementi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Body Weight ◽  
Influenza Virus ◽  
Influenza A ◽  
Human Monoclonal Antibody ◽  
Influenza A Viruses ◽  
Virus Challenge ◽  
Influenza A H1n1

ABSTRACTPN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011,http://dx.doi.org/10.1371/journal.pone.0028001). Previousin vitrostudies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypesin vitrobut also, more importantly, protect from a lethal influenza virus challengein vivo.

scholarly journals Favipiravir-resistant influenza A virus shows potential for transmission

2020 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Ada Yan ◽  
Rebecca Frise ◽  
Jie Zhou ◽  
Jennifer Shelley ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Genetic Background ◽  
Influenza A ◽  
Resistance Mutation ◽  
Polymerase Activity ◽  
Resistant Virus ◽  
Wild Type ◽  
Fitness Advantage

AbstractFavipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

scholarly journals Deletion of the Gene for the Type I Interferon Inhibitor I329L from the Attenuated African Swine Fever Virus OURT88/3 Strain Reduces Protection Induced in Pigs

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 262 ◽  
Author(s):  
Ana Luisa Reis ◽  
Lynnette C. Goatley ◽  
Tamara Jabbar ◽  
Elisabeth Lopez ◽  
Anusyah Rathakrishnan ◽  
...  
Keyword(s):  
Type I Interferon ◽  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Type I ◽  
Lethal Challenge ◽  
Host Innate Immune Response ◽  
Antibody Levels

Live attenuated vaccines are considered to be the fastest route to the development of a safe and efficacious African swine fever (ASF) vaccine. Infection with the naturally attenuated OURT88/3 strain induces protection against challenge with virulent isolates from the same or closely related genotypes. However, adverse clinical signs following immunisation have been observed. Here, we attempted to increase the OURT88/3 safety profile by deleting I329L, a gene previously shown to inhibit the host innate immune response. The resulting virus, OURT88/3ΔI329L, was tested in vitro to evaluate the replication and expression of type I interferon (IFN) and in vivo by immunisation and lethal challenge experiments in pigs. No differences were observed regarding replication; however, increased amounts of both IFN-β and IFN-α were observed in macrophages infected with the deletion mutant virus. Unexpectedly, the deletion of I329L markedly reduced protection against challenge with the virulent OURT88/1 isolate. This was associated with a decrease in both antibody levels against VP72 and the number of IFN-γ-producing cells in the blood of non-protected animals. Furthermore, a significant increase in IL-10 levels in serum was observed in pigs immunised with OURT88/3ΔI329L following challenge. Interestingly, the deletion of the I329L gene failed to attenuate the virulent Georgia/2007 isolate.

scholarly journals TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

2020 ◽  
Vol 11 (12) ◽  
pp. 894-914
Author(s):  
Nan Sun ◽  
Li Jiang ◽  
Miaomiao Ye ◽  
Yihan Wang ◽  
Guangwen Wang ◽  
...  
Keyword(s):  
Influenza A ◽  
Viral Infections ◽  
Influenza Infection ◽  
Type I ◽  
Subsequent Formation ◽  
Antiviral Immune Response ◽  
Signaling Complex ◽  
Deficient Mice

AbstractTripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.

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