Abstract
Background
Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low ...barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence.
Methods
Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR.
Results
I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments.
Conclusion
The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.
The I38T PA substitution associated with baloxavir resistance did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses in vitro. Similarly, the wild-type and its I38T mutant induced similar weight loss with comparable lung titers in a mouse model.
Background. Neuraminidase inhibitors (NAIs) play a key role in the management of influenza epidemics and pandemics. Given the novel pandemic influenza A(H1N1) (pH1N1) virus and the restricted number ...of approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority. Methods. Recombinant pH1N1 viruses were generated by reverse genetics, expressing either the wild-type or any of 9 mutant neuraminidase (NA) proteins (N2 numbering: E119G, E119V, D198G, 1222V, H274Y, N294S, S334N, I222V-H274Y, and H274Y-S334N). We evaluated these recombinant viruses for their resistance phenotype to 4 NAIs (oseltamivir, zanamivir, peramivir, and A-315675), NA enzymatic activity, and replicative capacity. Results. The E119G and E119V mutations conferred a multidrug resistance phenotype to many NAIs but severely compromised viral fitness. The oseltamivir- and peramivir-resistance phenotype was confirmed for the H274Y and N294S mutants, although both viruses remained susceptible to zanamivir. Remarkably, the I222V mutation had a synergistic effect on the oseltamivir- and peramivir-resistance phenotype of H274Y and compensated for reduced viral fitness, raising concerns about the potential emergence and dissemination of this double-mutant virus. Conclusions. This study highlights the importance of continuous monitoring of antiviral drug resistance in clinical samples as well as the need to develop new agents and combination strategies.
Baloxavir marboxil (BXM) is a potent inhibitor of the polymerase acidic (PA) protein of influenza viruses. However, clinical trials predominantly involving influenza A(H1N1) and A(H3N2) infections ...showed that BXM exhibited a low barrier of resistance. Contrasting with influenza A viruses, BXM-resistant influenza B variants remain poorly documented. We evaluated the impact of I38 T/M and E23K PA substitutions, previously reported in influenza A viruses, on in vitro properties and virulence of contemporary influenza B recombinant viruses. Influenza B/Phuket/3073/2013 recombinant wild-type (WT) virus and the I38T, I38M and E23K PA mutants were assessed for their susceptibility to baloxavir acid (BXA), the active metabolite of BXM, by plaque reduction assays in ST6GalI-MDCK cells. Luciferase-based minigenome tests were performed to determine polymerase activity. Replication kinetics and genetic stability were evaluated in ST6GalI-MDCK cells. Virulence was evaluated in BALB/c mice. The I38T, I38M and E23K substitutions increased BXA IC50s values by 12.6-, 5.5-, and 2.6-fold, respectively, compared to the WT. Minigenome assays revealed a 46% loss of polymerase activity for the E23K substitution vs the WT while the I38T and I38M PA variants retained ≈80% of activity. Peak viral titers were comparable for the WT, I38T and I38M recombinants (7.95 ± 0.5, 7.45 ± 0.25 and 8.11 ± 0.28 logTCID50/mL), respectively, whereas it was significantly lower for the E23K mutant (6.28 ± 0.28 logTCID50/mL;P < 0.05 vs the WT). In mice, the WT, I38T and I38M recombinants induced mortality rates of 60%, 40% and 100%, respectively and similar lung viral titers were obtained for the three groups at days 3 and 6 p.i. In conclusion, the fitness of BXA-resistant I38T and I38M PA mutants appears unaltered in contemporary influenza B viruses warranting surveillance for their emergence.
•Clinical trials showed that I38 T/M/F PA substitutions rapidly emerged in influenza A viruses causing baloxavir resistance.•We rescued B/Phuket/3073/2013 viruses with I38 T/M and E23K PA mutationsfor in vitro and mouse characterizations.•The I38T, I38M and E23K substitutions increased baloxavir IC50s by 12.6-, 5.5-, and 2.6-fold, respectively, vs the WT.•The I38 T/M mutants showed similar viral fitness vs the WT both in vitro and in mouse experiments.•The potential for baloxavir-resistance in influenza A and B viruses reinforces the need of novel antiviral strategies.
These infections are associated with a variety of clinical syndromes, in part related to specific serotype.
A new reverse transcription–polymerase chain reaction assay was developed for ...identification of 28 Canadian human parechovirus (HPeV) isolates, including 20 HPeV-1, 3 HPeV-2, and 5 HPeV-3, recovered from 1985 to 2004. All HPeV-1 isolates but 1 were genetically distinct from the Harris reference strain. One HPeV-2 isolate was related to the Williamson strain; the other 2 were related to the Connecticut strain. HPeV-3 isolates clustered together. Seventy-five percent of isolates were recovered during the typical enterovirus season. All patients but 1 were children with a mean age of 14.6 months, 6.3 months, and 0.7 months for HPeV-1, HPeV-2, and HPeV-3 patients, respectively. All HPeV-2– and HPeV-3–infected children were hospitalized with a diagnosis of viremia or sepsis. HPeV-1–infected children had bronchiolitis diagnosed in 50% of the cases, with few cases of pneumonia and enteritis. Two infected patients (1 child with leukemia and a 78-year-old woman) died of septic shock and severe pneumonia, respectively.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In Canada, cardiovirus isolates related to Saffold virus were detected in nasopharyngeal aspirates from 3 children with respiratory symptoms. Polyprotein sequence of the Can112051-06 isolate had ...91.2% aa identity with Saffold virus; however, EF and CD loops of the viral surface varied substantially.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 ...H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 µM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 µM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background. Development of influenza drug resistance is an important problem in immunocompromised children that could result in treatment failure and viral transmission to others. Methods.A total of ...17 influenza A/H3N2 isolates were recovered over a period of 1 year from an immunocompromised child who was initially treated with oseltamivir and then with amantadine and zanamivir for viral pneumonitis. Drug susceptibility phenotypes to oseltamivir, zanamivir, and peramivir were evaluated by neuraminidase (NA) inhibition assays, and sequence analysis of key viral genes (i.e., M2, NA, and hemagglutinin HA) was performed. The impact of NA mutations identified in oseltamivir-resistant isolates was analyzed using recombinant NA proteins. Results. An influenza A variant with NA mutations E59G, E119V, and I222V was first detected after 38 days of oseltamivir treatment. In an NA inhibition assay, this variant was 274 times more resistant to oseltamivir than the original isolate but was susceptible to zanamivir. The I222V substitution enhanced the level of oseltamivir resistance that was primarily conferred by the E119V mutation in recombinant NA proteins. Remarkably, the E119V mutation persisted for 8 months after cessation of oseltamivir. Amantadine therapy led to rapid emergence of the M2 mutation S31N, which is known to confer amantadine resistance. The patient shed the virus intermittently while receiving nebulized zanamivir therapy despite the absence of a resistance phenotype, which could be the result of nonoptimal drug delivery and impaired host immunity. Conclusions. This study highlights the potential for emergence and persistence of multidrug-resistant influenza isolates in immunocompromised subjects even after cessation of treatment, reinforcing the need for development of new anti-influenza compounds.
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