scholarly journals Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Jeremy C. Jones ◽  
Gyanendra Kumar ◽  
Subrata Barman ◽  
Isabel Najera ◽  
Stephen W. White ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Fold Change ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Next Generation ◽  
Influenza A H1n1

ABSTRACT The clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC 50 ]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC 50 ). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PA N target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro . Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs. IMPORTANCE The effectiveness of antiviral drugs can be severely compromised by the emergence of resistant viruses. Therefore, determination of the mechanisms by which viruses become resistant is critical for drug development and clinical use. RO-7 is a compound that potently inhibits influenza virus replication and belongs to a new class of drugs in late-stage clinical trials for treatment of influenza virus infection. Here we demonstrate that a single amino acid change acquired under prolonged virus exposure to RO-7 renders influenza viruses significantly less susceptible to its inhibitory effects. We have discovered how the mutation can simultaneously interfere with drug activity and still maintain efficient virus replication. These findings have important implications for the development of more effective derivatives of RO-7-like drugs and provide guidance for how to monitor the emergence of resistance.

scholarly journals Generation and Characterization of Recombinant Influenza A (H1N1) Viruses Harboring Amantadine Resistance Mutations

2005 ◽  
Vol 49 (2) ◽  
pp. 556-559 ◽  
Author(s):  
Yacine Abed ◽  
Nathalie Goyette ◽  
Guy Boivin
Keyword(s):  
Influenza A ◽  
Transmembrane Domain ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Resistance Mutations ◽  
Influenza A Viruses ◽  
Drug Concentrations ◽  
Influenza A H1n1 ◽  
Amantadine Resistance

ABSTRACT The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the matrix-(M)-2 protein. In this study, reverse genetics was used to generate and characterize recombinant influenza A (H1N1) viruses harboring L26F, V27A, A30T, S31N, G34E, and V27A/S31N mutations in the M2 gene. In plaque reduction assays, all mutations conferred amantadine resistance, with drug concentrations resulting in reduction of plaque number by 50% (IC50s) 154- to 3,300-fold higher than those seen for the wild type (WT). M2 mutants had no impairment in their replicative capacities in vitro on the basis of plaque size and replication kinetics experiments. In addition, all mutants were at least as virulent as the WT in experimentally infected mice, with the highest mortality rate being obtained with the recombinant harboring a double V27A/S31N mutation. These findings could help explain the frequent emergence and transmission of amantadine-resistant influenza viruses during antiviral pressure in the clinical setting.

scholarly journals Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James D. Allen ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Next Generation ◽  
Virus Infections ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

scholarly journals Next Generation of Computationally Optimized Broadly Reactive HA Vaccines Elicited Cross-Reactive Immune Responses and Provided Protection against H1N1 Virus Infection

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 793
Author(s):  
Ying Huang ◽  
Monique S. França ◽  
James D. Allen ◽  
Hua Shi ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Immune Responses ◽  
H1n1 Virus ◽  
Influenza Virus Infection ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Next Generation ◽  
Wild Type ◽  
Influenza A H1n1

Vaccination is the best way to prevent influenza virus infections, but the diversity of antigenically distinct isolates is a persistent challenge for vaccine development. In order to conquer the antigenic variability and improve influenza virus vaccine efficacy, our research group has developed computationally optimized broadly reactive antigens (COBRAs) in the form of recombinant hemagglutinins (rHAs) to elicit broader immune responses. However, previous COBRA H1N1 vaccines do not elicit immune responses that neutralize H1N1 virus strains in circulation during the recent years. In order to update our COBRA vaccine, two new candidate COBRA HA vaccines, Y2 and Y4, were generated using a new seasonal-based COBRA methodology derived from H1N1 isolates that circulated during 2013–2019. In this study, the effectiveness of COBRA Y2 and Y4 vaccines were evaluated in mice, and the elicited immune responses were compared to those generated by historical H1 COBRA HA and wild-type H1N1 HA vaccines. Mice vaccinated with the next generation COBRA HA vaccines effectively protected against morbidity and mortality after infection with H1N1 influenza viruses. The antibodies elicited by the COBRA HA vaccines were highly cross-reactive with influenza A (H1N1) pdm09-like viruses isolated from 2009 to 2021, especially with the most recent circulating viruses from 2019 to 2021. Furthermore, viral loads in lungs of mice vaccinated with Y2 and Y4 were dramatically reduced to low or undetectable levels, resulting in minimal lung injury compared to wild-type HA vaccines following H1N1 influenza virus infection.

scholarly journals Influenza: An Emerging and Re-emerging Public Health Threat in Nepal

2018 ◽  
Vol 3 (2) ◽  
pp. 1-2
Author(s):  
Bishnu Prasad Upadhyay
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Winter Season ◽  
Emerging Infectious Diseases ◽  
Influenza Viruses ◽  
Influenza B ◽  
Influenza A Viruses ◽  
Influenza A H1n1 ◽  
New Variant ◽  
Seasonal Epidemics

Influenza virus type A and B are responsible for seasonal epidemics as well as pandemics in human. Influenza A viruses are further divided into two major groups namely, low pathogenic seasonal influenza (A/H1N1, A/H1N1 pdm09, A/H3N2) and highly pathogenic influenza virus (H5N1, H5N6, H7N9) on the basis of two surface antigens: hemagglutinin (HA) and neuraminidase (NA). Mutations, including substitutions, deletions, and insertions, are one of the most important mechanisms for producing new variant of influenza viruses. During the last 30 years; more than 50 viral threat has been evolved in South-East Asian countriesof them influenza is one of the major emerging and re-emerging infectious diseases of global concern. Similar to tropical and sub-tropical countries of Southeast Asia; circulation of A/H1N1 pdm09, A/H3N2 and influenza B has been circulating throughout the year with the peak during July-November in Nepal. However; the rate of infection transmission reach peak during the post-rain and winter season of Nepal.

scholarly journals Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

2014 ◽  
Vol 95 (5) ◽  
pp. 1033-1042 ◽  
Author(s):  
Blanca García-Barreno ◽  
Teresa Delgado ◽  
Sonia Benito ◽  
Inmaculada Casas ◽  
Francisco Pozo ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A ◽  
Point Mutations ◽  
Antigenic Site ◽  
Single Amino Acid ◽  
Antigenic Structure ◽  
Human Antibody ◽  
2009 H1n1

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283–6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

scholarly journals Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

2008 ◽  
Vol 82 (14) ◽  
pp. 6902-6910 ◽  
Author(s):  
Frank T. Vreede ◽  
Hugh Gifford ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Influenza A ◽  
De Novo ◽  
Nucleoside Triphosphate ◽  
Ribonucleoprotein Complexes ◽  
Influenza Virus Replication ◽  
High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

scholarly journals N-linked glycosylation plays an important role in budding of NA protein and virulence of influenza viruses

2020 ◽  
Author(s):  
Danqi Bao ◽  
Ruixue Xue ◽  
Min Zhang ◽  
Chenyang Lu ◽  
Tianxin Ma ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Immune Escape ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
H1n1 Influenza Virus ◽  
Protein Loss ◽  
Knock Out

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of Hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knock-out significantly inhibited the budding of NA. Further studies showed that the NLG knock-out caused attenuation of virus in vitro and in vivo. Notably the NLG at 219 position plays an important role in budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, unfolded protein response (UPR) was determined in NLG knock-out NA overexpressed cells, which showed that the mutant NA was mainly located in ER, and the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knock-out NA was stacked in ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. Especially the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

scholarly journals Antiviral Activity of Aspalathus linearis against Human Influenza Virus

2017 ◽  
Vol 12 (4) ◽  
pp. 1934578X1701200 ◽  
Author(s):  
Ratika Rahmasari ◽  
Takahiro Haruyama ◽  
Siriwan Charyasriwong ◽  
Tomoki Nishida ◽  
Nobuyuki Kobayashi
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Time Course ◽  
Influenza Viruses ◽  
Replication Process ◽  
Human Influenza Virus ◽  
Alkaline Extract ◽  
Aspalathus Linearis

Influenza A viruses are responsible for annual epidemics and occasional pandemics, which cause significant morbidity and mortality. The limited protection offered by influenza vaccination, and the emergence of drug-resistant influenza strains, highlight the urgent need for the development of novel anti-influenza drugs. However, the search for antiviral substances from the library of low molecular weight chemical compounds is limited. Thus, because of their natural diversity and accessibility, plants or plant-derived materials are rapidly becoming valuable sources for the discovery and development of new antiviral drugs. In this study, crude extracts of Aspalathus linearis, a plant reported to have anti-HIV activity, were evaluated in vitro for their activity against the influenza A virus. Of the extracts tested, an alkaline extract of Aspalathus linearis demonstrated the strongest inhibition against influenza A virus and could also inhibit different types of influenza viruses, including Oseltamivir-resistant influenza viruses A and B. Our time course of addition studies indicated that the alkaline extract of Aspalathus linearis exerts its antiviral effect predominantly during the late stages of the influenza virus replication process.

scholarly journals Molecular Epidemiology and Antigenic Characterization of Seasonal Influenza Viruses Circulating in Nepal

2017 ◽  
Vol 15 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Bishu Prasad Upadhyay ◽  
Prakash Ghimire ◽  
Masato Tashiro ◽  
Mogha Raj Banjara
Keyword(s):  
New York ◽  
Influenza Virus ◽  
Molecular Epidemiology ◽  
Influenza A ◽  
Influenza Viruses ◽  
Influenza B ◽  
Antigenic Characterization ◽  
Geographical Regions ◽  
Influenza A H1n1 ◽  
Health Burdens

Background: Influenza is one of the public health burdens in Nepal and its epidemiology is not clearly understood. The objective of this study was to explore the molecular epidemiology and the antigenic characteristics of the circulating influenza viruses in Nepal.Methods: A total of 1495 throat swab specimens were collected from January to December, 2014. Real time PCR assay was used for identification of influenza virus types and subtypes. Ten percent of the positive specimens were randomly selected and inoculated onto Madin-Darby Canine Kidney Epithelial cells (MDCK) for influenza virus isolation. All viruses were characterized by the hemagglutination inhibition (HI) assay.Results: Influenza viruses were detected in 421/1495 (28.2%) specimens. Among positive cases, influenza A virus was detected in 301/421 (71.5%); of which 120 (39.9%) were influenza A/H1N1 pdm09 and 181 (60.1%) were influenza A/H3 subtype. Influenza B viruses were detected in 119/421 (28.3%) specimens. Influenza A/H1N1 pdm09, A/H3 and B viruses isolated in Nepal were antigenically similar to the vaccine strain influenza A/California/07/2009(H1N1pdm09), A/Texas/50/2012(H3N2), A/New York/39/2012(H3N2) and B/Massachusetts/2/2012, respectively.Conclusions: Influenza viruses were reported year-round in different geographical regions of Nepal which was similar to other tropical countries. The circulating influenza virus type and subtypes of Nepal were similar to vaccine candidate virus which could be prevented by currently used influenza vaccine.

scholarly journals Contemporary Seasonal Influenza A (H1N1) Virus Infection Primes for a More Robust Response To Split Inactivated Pandemic Influenza A (H1N1) Virus Vaccination in Ferrets

2010 ◽  
Vol 17 (12) ◽  
pp. 1998-2006 ◽  
Author(s):  
Ali H. Ellebedy ◽  
Thomas P. Fabrizio ◽  
Ghazi Kayali ◽  
Thomas H. Oguin ◽  
Scott A. Brown ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Seasonal Influenza ◽  
H1n1 Virus ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Inactivated Vaccine ◽  
H1n1 Influenza Virus ◽  
Influenza A H1n1

ABSTRACT Human influenza pandemics occur when influenza viruses to which the population has little or no immunity emerge and acquire the ability to achieve human-to-human transmission. In April 2009, cases of a novel H1N1 influenza virus in children in the southwestern United States were reported. It was retrospectively shown that these cases represented the spread of this virus from an ongoing outbreak in Mexico. The emergence of the pandemic led to a number of national vaccination programs. Surprisingly, early human clinical trial data have shown that a single dose of nonadjuvanted pandemic influenza A (H1N1) 2009 monovalent inactivated vaccine (pMIV) has led to a seroprotective response in a majority of individuals, despite earlier studies showing a lack of cross-reactivity between seasonal and pandemic H1N1 viruses. Here we show that previous exposure to a contemporary seasonal H1N1 influenza virus and to a lesser degree a seasonal influenza virus trivalent inactivated vaccine is able to prime for a higher antibody response after a subsequent dose of pMIV in ferrets. The more protective response was partially dependent on the presence of CD8+ cells. Two doses of pMIV were also able to induce a detectable antibody response that provided protection from subsequent challenge. These data show that previous infection with seasonal H1N1 influenza viruses likely explains the requirement for only a single dose of pMIV in adults and that vaccination campaigns with the current pandemic influenza vaccines should reduce viral burden and disease severity in humans.

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