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Publications (134)
Antiviral susceptibility of influenza viruses was assessed using a high-content imaging-based neutralization test. Cap-dependent endonuclease inhibitors, baloxavir and AV5116, were superior to AV5115 against type A viruses, and AV5116 was most effective against PA mutants tested. However, these three inhibitors displayed comparable activity (EC50 8...
Clade 2.3.4.4 b highly pathogenic avian influenza (HPAI) A (H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1015 sequences of HPAI A (H5N1) viruses collected in the United States during 2022, eight viruses...
Year-round virological characterization of circulating epidemic influenza viruses is conducted worldwide to detect the emergence of viruses that may escape pre-existing immunity or acquire resistance to antivirals. High throughput phenotypic assays are needed to complement the sequence-based analysis of circulating viruses and improve pandemic prep...
Four cases of oseltamivir-resistant influenza A(H1N1)pdm09 virus infection were detected among inhabitants of a border detention center in Texas, USA. Hemagglutinin of these viruses belongs to 6B.1A5A-156K subclade, which may enable viral escape from preexisting immunity. Our finding highlights the necessity to monitor both drug resistance and anti...
Pimodivir exerts an antiviral effect on the early stages of influenza A virus replication by inhibiting the cap-binding function of polymerase basic protein 2 (PB2). In this study, we used a combination of sequence analysis and phenotypic methods to evaluate pimodivir susceptibility of influenza A viruses collected from humans and other hosts. Scre...
Baloxavir, a new antiviral drug targeting cap-dependent endonuclease activity of polymerase acidic (PA) protein of influenza viruses, is now approved in multiple countries. Several substitutions at isoleucine 38 in PA protein (e.g., PA-I38T) have been associated with decreased baloxavir susceptibility in vitro and in vivo. In recent years, next gen...
Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A ˃ B ˃ C ˃ D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neurami...
Recently circulating seasonal influenza A viruses carrying PA-I38L/M/S/T substitutions showed differential effect on baloxavir susceptibility and in vitro replicative fitness. In ferret infection model, replication of A(H3N2) viruses carrying I38M or I38T was mildly impaired compared to control/wild type viruses. Abstract Susceptibility of influenz...
Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold), but...
Mutations in the influenza virus neuraminidase (NA) that cause reduced susceptibility to the NA inhibitor (NAI) oseltamivir may occur naturally or following antiviral treatment. Currently, detection uses either a traditional NA inhibition assay or gene sequencing to identify known markers associated with reduced inhibition by oseltamivir. Both meth...
Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using haemagglutination inhibition and promote selection of HA mutants. Thus, alternative approaches are needed to reliably ass...
The anti-influenza therapeutic baloxavir targets cap-dependent endonuclease activity of polymerase acidic (PA) protein. We monitored baloxavir susceptibility in the United States with next generation sequencing analysis supplemented by phenotypic one-cycle infection assay. Analysis of PA sequences of 6,891 influenza A and B viruses collected during...
A new rapid assay for detecting oseltamivir resistance in influenza virus, iART, was used to test 149 clinical specimens. Results were obtained for 132, with iART indicating 41 as ‘resistant’. For these, sequence analysis found known and suspected markers of oseltamivir resistance, while no such markers were detected for the remaining 91 samples. V...
In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused...
Quantification of a proportion of H134 and N134 neuraminidase variants in respiritory specimens harboing influenza B viruses collected in Laos, February 2016.
Mounting evidence suggests that neuraminidase’s functionality extends beyond its classical role in influenza virus infection and that anti-neuraminidase antibodies offer protective immunity. Therefore, a renewed interest in development of neuraminidase (NA)-specific methods to characterize the glycoprotein and evaluate potential advantages for NA s...
Rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain sele...
Importance:
Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here we demonstrate that the hemagglutin...
During 2014, a subclade 2.3.4.4 HPAI A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015 the virus was detected in wild birds in Canada and the U.S. and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North America lineages. In particular, viruses were f...
Background:
During the 2014-15 US influenza season, expanded genetic characterization of circulating influenza A(H3N2) viruses was used to assess the impact of genetic variability of influenza A(H3N2) viruses on influenza vaccine effectiveness (VE).
Methods:
A novel pyrosequencing assay was used to determine genetic group based on hemagglutinin...
Background:
Monitoring influenza virus susceptibility to neuraminidase (NA) inhibitors (NAIs) is vital for detecting drug-resistant variants, and is primarily assessed using NA inhibition (NI) assays, supplemented by NA sequence analysis. However, differences in NI testing methodologies between surveillance laboratories results in variability of 5...
Preliminary results for influenza vaccine effectiveness (VE) against acute respiratory illness with circulating laboratory-confirmed influenza viruses in New Zealand from 27 April to 26 September 2015, using a case testnegative design were 36% (95% confidence interval (CI): 11–54) for general practice encounters and 50% (95% CI: 20–68) for hospital...
Prolonged treatment of an immunocompromised child with oseltamivir and zanamivir for A(H1N1)pdm09 virus infection led to the emergence of viruses carrying H275Y and/or E119G in the neuraminidase. When phenotypically evaluated by neuraminidase inhibition, the dual H275Y-E119G substitution caused highly reduced inhibition by four neuraminidase inhibi...
Unlabelled:
Human infections by avian influenza A(H7N9) virus entail substantial morbidity and mortality. Treatment of infected patients with the neuraminidase (NA) inhibitor oseltamivir was associated with emergence of viruses carrying NA substitutions. In the NA inhibition (NI) assay, R292K conferred highly reduced inhibition by oseltamivir, whi...
National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis; and pyrosequencing analysis of clinical specimens. Lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay d...
Background:
Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for influenza A(H7N9) is limited.
Methods:
Four NA variants of A/Taiwan/1/2013(H7N9) virus containing a sing...
Since 2011, outbreaks caused by influenza A(H3N2) variant [A(H3N2)v] viruses have become a public health concern in the United States. The A(H3N2)v viruses share the A(H1N1)pdm09 M gene containing the marker of M2 blocker resistance, S31N, but do not contain any known molecular markers associated with resistance to neuraminidase (NA) inhibitors (NA...
Human infections caused by avian influenza A virus type subtype H7N9 have been associated with substantial morbidity and mortality.
Emergence of virus variants carrying markers of decreased susceptibility to neuraminidase inhibitors was reported. Here we
show that DAS181 (Fludase), an antiviral drug with sialidase activity, potently inhibited repli...
We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009-2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inh...
Drug susceptibility and accession data for highly pathogenic avian influenza A(H5N1) viruses.
Analysis of highly pathogenic avian influenza A(H5N1) viruses.
Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ∼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of t...
Assessment 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-grown isolate of this virus exhibited highly reduced inhibit...
The QFlu™ prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point-of-care. Here we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n=14) and a set of 90 seasonal i...
Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. H...
HA mutants that determine a decrease of PN-SIA49 binding to HA are expressed at the same level of wild type HA on cell surface. FACS curves showing the binding of anti-H1N1 HA antibody (directed against a linear epitope) to untransfected cells, HA wild-type and HA-mutants. White and red curves represent, for each graph, respectively the binding of...
During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir trea...
Neutralization assays against viruses from group 1 subtypes. Dose–response curve fit nonlinear regression is reported for PN-SIA28 against H1N1, H5N1, H2N2 and H9N2 strains studied in this paper. Data from two different experiments for each strain are reported. Each point was performed in triplicate.
(TIF)
Unrooted tree of HA1 region of H3 hemagglutinins. The different isolates belong to different historical periods spanning 1968 to 2009. The three different clusters characterizing the H3N2 pandemic are evidenced, with each cluster including isolates belonging to a distinct time period of the pandemic. Green ‘+’ or red ‘−’ indicate PN-SIA28 positive...
Influenza hemagglutinin unrooted phylogenetic tree of all the viral strains tested in neutralization assays with PN-SIA49. Viral isolates belonging to group 1 and group 2 are divided into two different boxes. Subtypes that can be neutralized by PN-SIA49 are indicated with a green ‘+’, while the ones that cannot be neutralized are indicate with a re...
HA amino acidic residues mutated in H1 and H3.
(DOC)
The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not...
The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that...
[This corrects the article on p. 2269 in vol. 2.].
Antiviral drugs play an essential role in managing infections caused by seasonal and pandemic influenza viruses. Due to the high prevalence of damantine resistance among seasonal influenza A viruses circulating in certain geographic regions, neuraminidase inhibitors (NAIs) are presently the only effective antiviral drugs for treatment and chemoprop...
For influenza viruses, pyrosequencing has been successfully applied to the high-throughput detection of resistance markers
in genes encoding the drug-targeted M2 protein and neuraminidase. In this study, we expanded the utility of this assay to
the detection of multiple receptor binding variants of the hemagglutinin protein of influenza viruses dir...
Patient's characteristics and outcomes according to age range.
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Prevalence of underlying malignancies.
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Bacteria isolates from cancer patients with Influenza A H1N1pdm.
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Organ Dysfunctions 72 h after H1N1pdm diagnosis.
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Clinical investigation and Influenza virus assays.
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Diagnostic tests for Influenza A virus.
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Oseltamivir treatments.
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Focus of bacterial infection in cancer patients with Influenza A H1N1pdm.
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Phylogenetic tree of NA gene from the followed-up cohort. The bootstrap probability is not indicated for each interior branch since it is below 85%. The scale bar indicates the number of amino acid changes per site. The tree is rooted by California/07/2009 NA sequence.
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Use of Chemotherapy, corticosteroids and granulocyte colony stimulating factor previous to H1N1pdm infection.
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Patient's Neutropenia.
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Frequency of signs and symptoms at clinical suspicion.
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Pulmonary infiltrates at Influenza diagnosis and hospital discharge, by chest radiography.
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Clinical and Viral Characteristics of the followed-up cohort.
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Phylogenetic tree of HA gene from the classical swine, Eurasian swine, American Avian and human seasonal lineages. The bootstrap probability is indicated for each interior branch, all values below 80% are hidden. The scale bar indicates the number of amino acid changes per site. Colored circles indicate the samples from our study. This tree is unro...
The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 i...
Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010.
Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adama...
Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008-2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC(50)s were performed for eac...
Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA
viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment
of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its abil...
The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second rec...
The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibi...
Background:
The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread os...
The human parainfluenza viruses (hPIVs) and respiratory syncytial viruses (RSVs) are the leading causes of hospitalizations due to respiratory viral disease in infants and young children, but no vaccines are yet available. Here we describe the use of recombinant Sendai viruses (rSeVs) as candidate vaccine vectors for these respiratory viruses in a...
Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects young children, but no vaccines or antiviral drugs against it have yet been developed. We developed a mouse model to evaluate the efficacies of the novel parainfluenza virus hemagglutinin-neuraminidase (HN) inhibitors BCX 2798 and BCX 2855 against a recombi...
Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation s...
BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-...
Influenza A/New Caledonia/20/99 (H1N1) virus was studied for development of resistance to cyanovirin-N (CVN). CVN neutralizes virus infectivity by binding to specific high-mannose oligosaccharides on the viral haemagglutinin 1 (HA1) subunit. During virus adaptation to mice in the absence of CVN treatment the virus became resistant to CVN (CVN-MR vi...
An inbred murine model (BALB/c) was utilized to assess the protective effect of the immunomodulator methyl inosine 5'-monophosphate (MIMP) against infection with influenza A/PR/8/34 (H1N1) virus. Contrary to the data reported for outbred mice (NMRI) infected with the aerosolized virus (Masihi, Hadden, 2003. J. Int. Immunopharmacol. 3, 1205-1215), t...
We developed an immunocompromised murine model of influenza virus infection and demonstrated comparable efficacy of oral oseltamivir
and A-322278 (both given at dosages of 10 mg/kg/day) in reducing viral replication, decreasing weight loss, and prolonging
survival. Once the treatment was discontinued, severe combined immunodeficient (SCID) mice had...
Influenza is a highly infectious disease characterized by recurrent annual epidemics and unpredictable major worldwide pandemics.
Rapid spread of the highly pathogenic avian H5N1 strain and escalating human infections by the virus have set off the alarm
for a global pandemic. To provide an urgently needed alternative treatment modality for influenz...
Protective efficacy of the intranasal immunization with the neuraminidase (NA)-deficient mutant of the influenza A virus was investigated in ferrets. Despite the highly attenuated replication in vivo, the mutant completely protected the animals against the wild type virus challenge. When challenge was done with antigenic drift variants, significant...
The susceptibilities of five zanamivir-resistant and six oseltamivir-resistant influenza viruses were assessed against four
neuraminidase (NA) inhibitors, including peramivir and A-315675, by a fluorometric NA activity inhibition assay. The enzyme
activity of a majority of the variants was effectively inhibited by either A-315675 or both peramivir...
Inhibition of neuraminidase (NA) activity prevents release of progeny virions from influenza-infected cells and removal of
neuraminic (sialic) acid moieties from glycans attached to hemagglutinin (HA). Neuraminic acid moieties situated near the
HA receptor-binding site can reduce the efficiency of virus binding and decrease viral dependence on NA a...
In a short time, West Nile virus has developed into a nationwide health and veterinary problem. The high virulence of the circulating virus and related lineage 1 WN strains hinders development of an attenuated live vaccine. We describe an attenuated WN isolate, WN1415, which is a molecularly cloned descendant of the WN prototype B956 strain. The pa...
