Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models

AbstractMost anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

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Interferon Lambda Delays the Emergence of Influenza Virus Resistance to Oseltamivir

Microorganisms ◽

10.3390/microorganisms9061196 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1196

Author(s):

Chiara Medaglia ◽

Arnaud Charles-Antoine Zwygart ◽

Paulo Jacob Silva ◽

Samuel Constant ◽

Song Huang ◽

...

Keyword(s):

Influenza Virus ◽

Neuraminidase Inhibitor ◽

Influenza Viruses ◽

Human Interferon ◽

Infection Model ◽

Single Mutation ◽

Resistance Mutations ◽

Species Barrier ◽

Interferon Lambda

Influenza viruses are a leading cause of morbidity and mortality worldwide. These air-borne pathogens are able to cross the species barrier, leading to regular seasonal epidemics and sporadic pandemics. Influenza viruses also possess a high genetic variability, which allows for the acquisition of resistance mutations to antivirals. Combination therapies with two or more drugs targeting different mechanisms of viral replication have been considered an advantageous option to not only enhance the effectiveness of the individual treatments, but also reduce the likelihood of resistance emergence. Using an in vitro infection model, we assessed the barrier to viral resistance of a combination therapy with the neuraminidase inhibitor oseltamivir and human interferon lambda against the pandemic H1N1 A/Netherlands/602/2009 (H1N1pdm09) virus. We serially passaged the virus in a cell line derived from human bronchial epithelial cells in the presence or absence of increasing concentrations of oseltamivir alone or oseltamivir plus interferon lambda. While the treatment with oseltamivir alone quickly induced the emergence of antiviral resistance through a single mutation in the neuraminidase gene, the co-administration of interferon lambda delayed the emergence of drug-resistant influenza virus variants. Our results suggest a possible clinical application of interferon lambda in combination with oseltamivir to treat influenza.

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Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo

Scientific Reports ◽

10.1038/s41598-021-88886-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Eun-Jung Song ◽

Erica Españo ◽

Sang-Mu Shim ◽

Jeong-Hyun Nam ◽

Jiyeon Kim ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Lethal Dose ◽

Influenza Viruses ◽

Influenza B Virus ◽

Influenza B ◽

Inhibitory Effects ◽

Compound Libraries ◽

Wide Range

AbstractInfluenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

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Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

Medical academic journal ◽

10.17816/maj77563 ◽

2021 ◽

Vol 21 (3) ◽

pp. 153-158

Author(s):

Anna K. Chistyakova ◽

Polina I. Prokopenko ◽

Elena V. Krutikova ◽

Ekaterina A. Stepanova ◽

Irina N. Isakova-Sivak ◽

...

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Influenza A ◽

Influenza Viruses ◽

T Cell Epitopes ◽

Ns1 Protein ◽

Cold Adapted ◽

Ns Gene ◽

Wide Range

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion. AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro. MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos. Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature. CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

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Chaiqin Qingning Capsule Inhibits Influenza Virus Infection and Inflammation In Vitro and In Vivo

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/6640731 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Jin Zhao ◽

Yanbing Hao ◽

Xuanzi Xia ◽

Runfeng Li ◽

Xiaodong Huang ◽

...

Keyword(s):

Influenza Virus ◽

A549 Cells ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Dependent Manner ◽

Lung Pathology ◽

Chemokine Expression ◽

Wide Range

Background. Chaiqin Qingning Capsule (CQ-C) is a traditional Chinese medicine (TCM) formula commonly used to treat respiratory infectious diseases in China. The aim of this study was to detect the effect and mechanism of CQ-C treated with influenza virus in vitro and vivo. Methods. The cytotoxicity and antiviral activity of CQ-C in vitro was determined by methyl thiazolyl tetrazolium (MTT) assay. The regulation of CQ-C on cytokine/chemokine expression was evaluated using RT-qPCR. In addition, the effect of CQ-C on the pathway protein, NF-κB, and its phosphorylation level was verified by western blotting. After virus inoculation, BALB/c mice were administered with CQ-C of different concentrations for 7 days. Body weight, viral titer, lung pathology, and mortality of the mice were measured, and the level of inflammatory cytokines was also examined using real-time RT-qPCR. Results. CQ-C inhibited the proliferation of influenza virus of various strains in vitro, with the 50% inhibitory concentration (IC50) ranging from 49 to 59 µg/mL. CQ-C downregulated virus-induced gene expression of IL-6, TNF-α, CXCL8, CXCL10, CCL5, and COX-2 in a dose-dependent manner in A549 cells. Also, CQ-C inhibited the expression of NF-κB protein of the signaling pathway. Moreover, a decrease of the lung index and mortality of mice was observed in the CQ-C (1 g/kg/d) group. The related cytokine/chemokine expression was also decreased in the early stages of infection in the mRNA level. Conclusion. As a clinically applied Chinese prescription, our study shows that CQ-C has a wide range of effects on several influenza viruses. Moreover, CQ-C could play an important role in anti-influenza activity and anti-inflammation in vitro and in vivo. Thus, CQ-C may be a promising treatment option for influenza.

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Cetylpyridinium Chloride (CPC) Exhibits Potent, Rapid Activity Against Influenza Viruses in vitro and in vivo

Pathogens and Immunity ◽

10.20411/pai.v2i2.200 ◽

2017 ◽

Vol 2 (2) ◽

pp. 253 ◽

Cited By ~ 13

Author(s):

Daniel L. Popkin ◽

Sarah Zilka ◽

Matthew Dimaano ◽

Hisashi Fujioka ◽

Cristina Rackley ◽

...

Keyword(s):

Influenza Virus ◽

Antimicrobial Agent ◽

Prolonged Exposure ◽

Cetylpyridinium Chloride ◽

Influenza Viruses ◽

Viral Envelope ◽

Ammonium Compound ◽

Virucidal Activity ◽

Resistant Strains

AbstractBackground: There is a continued need for strategies to prevent influenza. While cetylpyridinium chloride (CPC), a broad-spectrum antimicrobial agent, has an extensive antimicrobial spectrum, its ability to affect respiratory viruses has not been studied in detail.Objectives: Here, we evaluate the ability of CPC to disrupt influenza viruses in vitro and in vivo.Methods: The virucidal activity of CPC was evaluated against susceptible and oseltamivir- resistant strains of influenza viruses. The effective virucidal concentration (EC) of CPC was determined using a hemagglutination assay and tissue culture infective dose assay. The effect of CPC on viral envelope morphology and ultrastructure was evaluated using transmission electron microscopy (TEM). The ability of influenza virus to develop resistance was evaluated after multiple passaging in sub-inhibitory concentrations of CPC. Finally, the efficacy of CPC in formulation to prevent and treat influenza infection was evaluated using the PR8 murine influenza model.Results: The virucidal effect of CPC occurred within 10 minutes, with mean EC50 and EC2log ranging between 5 to 20 µg/mL, for most strains of influenza tested regardless of type and resistance to oseltamivir. Examinations using TEM showed that CPC disrupted the integrity of the viral envelope and its morphology. Influenza viruses demonstrated no resistance to CPC despite prolonged exposure. Treated mice exhibited significantly increased survival and maintained body weight compared to untreated mice.Conclusions: The antimicrobial agent CPC possesses virucidal activity against susceptible and resistant strains of influenza virus by targeting and disrupting the viral envelope. Substantial virucidal activity is seen even at very low concentrations of CPC without development of resistance. Moreover, CPC in formulation reduces influenza-associated mortality and morbidity in vivo.Keywords: Cetylpyridinium Chloride, CPC, Influenza, Respiratory tract illness, respiratory virus, quaternary ammonium compound

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The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

Journal of Virology ◽

10.1128/jvi.73.2.1293-1301.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1293-1301 ◽

Cited By ~ 21

Author(s):

Kazunori Inabe ◽

Masako Nishizawa ◽

Shigeru Tajima ◽

Kazuyoshi Ikuta ◽

Yoko Aida

Keyword(s):

Viral Entry ◽

Envelope Protein ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Transient Transfection ◽

Viral Envelope ◽

Tyrosine Residue ◽

Wild Type ◽

Viral Envelope Protein

ABSTRACT The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

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Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.12.3234 ◽

1998 ◽

Vol 42 (12) ◽

pp. 3234-3241 ◽

Cited By ~ 108

Author(s):

Chun Y. Tai ◽

Paul A. Escarpe ◽

Robert W. Sidwell ◽

Matthew A. Williams ◽

Willard Lew ◽

...

Keyword(s):

Influenza Virus ◽

Neuraminidase Inhibitor ◽

H3n2 Virus ◽

Wild Type Virus ◽

Type Virus ◽

Human Influenza ◽

Wild Type ◽

Viral Virulence ◽

High Level

ABSTRACT An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

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Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00793-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4962-4973 ◽

Cited By ~ 32

Author(s):

Yasushi Itoh ◽

Shintaro Shichinohe ◽

Misako Nakayama ◽

Manabu Igarashi ◽

Akihiro Ishii ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Cynomolgus Macaque ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

H7n9 Virus ◽

Macaque Model ◽

H7n9 Influenza Virus ◽

Number Of Patients ◽

H7n9 Influenza

ABSTRACTThe number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitorsin vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.

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Characterization of the VP39 envelope protein from Singapore grouper iridovirus

Canadian Journal of Microbiology ◽

10.1139/cjm-2015-0118 ◽

2015 ◽

Vol 61 (12) ◽

pp. 924-937 ◽

Cited By ~ 8

Author(s):

Honglian Zhang ◽

Sheng Zhou ◽

Liqun Xia ◽

Xiaohong Huang ◽

Youhua Huang ◽

...

Keyword(s):

Envelope Protein ◽

Core Gene ◽

Immunofluorescence Assay ◽

Current Data ◽

Viral Envelope ◽

Economic Losses ◽

Immunogold Electron Microscopy ◽

Viral Envelope Protein ◽

Drug Inhibition

Singapore grouper iridovirus (SGIV) is a major pathogen that causes heavy economic losses to the grouper aquaculture industry in China and Southeast Asian countries. In the present study, a viral envelope protein, VP39, encoded by SGIV ORF39L, was identified and characterized. SGIV ORF39L was found in all sequenced iridoviruses and is now considered to be a core gene of the family Iridoviridae. ORF39L was classified as a late gene during in vitro infection using reverse transcription–polymerase chain reaction, western blotting, and a drug inhibition analysis. An indirect immunofluorescence assay revealed that the VP39 protein was confined to the cytoplasm, especially at viral assembly sites. Western blot and matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry analyses suggested that VP39 is an envelope protein. Immunogold electron microscopy further confirmed that VP39 is a viral envelope protein. Furthermore, a mouse anti-VP39 polyclonal antibody exhibited SGIV-neutralizing activity in vitro, suggesting that VP39 is involved in SGIV infection. Taken together, the current data suggest that VP39 represents a conserved envelope protein of iridoviruses that contributes to viral infection.

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Endoplasmic Reticulum Stress Is a Determinant of Retrovirus-Induced Spongiform Neurodegeneration

Journal of Virology ◽

10.1128/jvi.77.23.12617-12629.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12617-12629 ◽

Cited By ~ 59

Author(s):

Derek E. Dimcheff ◽

Srdjan Askovic ◽

Audrey H. Baker ◽

Cedar Johnson-Fowler ◽

John L. Portis

Keyword(s):

Endoplasmic Reticulum ◽

Transmissible Spongiform Encephalopathy ◽

Viral Envelope ◽

Spongiform Encephalopathy ◽

Envelope Gene ◽

Viral Envelope Protein ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT FrCasE is a mouse retrovirus that causes a fatal noninflammatory spongiform neurodegenerative disease with pathological features strikingly similar to those induced by transmissible spongiform encephalopathy (TSE) agents. Neurovirulence is determined by the sequence of the viral envelope protein, though the specific role of this protein in disease pathogenesis is not known. In the present study, we compared host gene expression in the brain stems of mice infected with either FrCasE or the avirulent virus F43, differing from FrCasE in the sequence of the envelope gene. Four of the 12 disease-specific transcripts up-regulated during the preclinical period represent responses linked to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Among these genes was CHOP/GADD153, which is induced in response to conditions that perturb endoplasmic reticulum function. In vitro studies with NIH 3T3 cells revealed up-regulation of CHOP as well as BiP, calreticulin, and Grp58/ERp57 in cells infected with FrCasE but not with F43. Immunoblot analysis of infected NIH 3T3 cells demonstrated the accumulation of uncleaved envelope precursor protein in FrCasE- but not F43-infected cells, consistent with ER retention. These results suggest that retrovirus-induced spongiform neurodegeneration represents a protein-folding disease and thus may provide a useful tool for exploring the causal link between protein misfolding and the cytopathology that it causes.

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