ABSTRACT
Replication of influenza A virus (IAV) from negative-sense viral RNA (vRNA) requires the generation of positive-sense RNA (+RNA). Most molecular assays, such as conventional real-time reverse transcriptase PCR (rRT-PCR), detect total RNA in a sample without differentiating vRNA from +RNA. These assays are not designed to distinguish IAV infection versus exposure of an individual to an environment enriched with IAVs but wherein no viral replication occurs. We therefore developed a strand-specific hybridization (SSH) assay that differentiates between vRNA and +RNA and quantifies relative levels of each RNA species. The SSH assay exhibited a linearity of 7 logs with a lower limit of detection of 6.0 × 102 copies of molecules per reaction. No signal was detected in samples with a high load of nontarget template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected 2 to 4 h postinoculation of MDCK cells, whereas synthesis of cold-adapted IAV +RNA was significantly impaired at 37°C. The SSH assay was then used to test IAV rRT-PCR positive nasopharyngeal specimens collected from individuals exposed to IAV at swine exhibitions (n = 7) or while working at live bird markets (n = 2). The SSH assay was able to differentiate vRNA and +RNA in samples collected from infected, symptomatic individuals versus individuals who were exposed to IAV in the environment but had no active viral replication. Data generated with this technique, especially when coupled with clinical data and assessment of seroconversion, will facilitate differentiation of actual IAV infection with replicating virus versus individuals exposed to high levels of environmental contamination but without virus infection.
Subject(s)
Influenza A virus , Influenza, Human , Animals , Dogs , Humans , Influenza A virus/genetics , Influenza, Human/diagnosis , Madin Darby Canine Kidney Cells , RNA, Viral/genetics , Swine , Virus ReplicationABSTRACT
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 Ë B Ë C Ë D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.
Subject(s)
Antiviral Agents/pharmacology , Gammainfluenzavirus/drug effects , Influenza A virus/drug effects , Influenza B virus/drug effects , Oxazines/pharmacology , Pyridines/pharmacology , Thiepins/pharmacology , Thogotovirus/drug effects , Triazines/pharmacology , Animals , Chickens/virology , Dibenzothiepins , Dogs , Humans , Influenza in Birds/drug therapy , Influenza in Birds/virology , Influenza, Human/drug therapy , Influenza, Human/virology , Madin Darby Canine Kidney Cells/virology , Microbial Sensitivity Tests , Morpholines , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/veterinary , Orthomyxoviridae Infections/virology , Pyridones , Swine/virology , Swine Diseases/drug therapy , Swine Diseases/virologyABSTRACT
Infections with low pathogenicity and highly pathogenic avian influenza A(H7N9) viruses affected poultry in 4 states in the southeastern United States in 2017. We evaluated pathogenicity and transmission of representative viruses in mouse and ferret models and examined replication kinetics in human respiratory tract cells. These viruses can cause respiratory infections in mammalian models.
Subject(s)
Influenza A Virus, H7N9 Subtype/pathogenicity , Influenza in Birds/virology , Orthomyxoviridae Infections/veterinary , Animals , Cell Line , Chickens/virology , Disease Outbreaks/veterinary , Ferrets/virology , Humans , Influenza in Birds/epidemiology , Influenza, Human/virology , Mice , Orthomyxoviridae Infections/virology , Respiratory System/cytology , Tennessee/epidemiology , VirulenceSubject(s)
Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Respiratory Tract Infections , Humans , Infant , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/prevention & controlABSTRACT
Background: Neuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse. Methods: Fourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed. Results: All substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir. Conclusions: The recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Multiple, Viral/genetics , Influenza A Virus, H7N9 Subtype/drug effects , Neuraminidase/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Acids, Carbocyclic , Cyclopentanes/pharmacology , Databases, Genetic , Enzyme Inhibitors/pharmacology , Guanidines/pharmacology , Humans , Influenza A Virus, H7N9 Subtype/genetics , Influenza, Human/drug therapy , Inhibitory Concentration 50 , Neuraminidase/genetics , Oseltamivir/pharmacology , Protein Conformation , Pyrans , Recombinant Proteins/genetics , Sialic Acids , Viral Proteins/genetics , Zanamivir/analogs & derivatives , Zanamivir/pharmacologyABSTRACT
An outbreak of influenza A(H7N2) virus in cats in a shelter in New York, NY, USA, resulted in zoonotic transmission. Virus isolated from the infected human was closely related to virus isolated from a cat; both were related to low pathogenicity avian influenza A(H7N2) viruses detected in the United States during the early 2000s.
Subject(s)
Cat Diseases/epidemiology , Disease Outbreaks , Genome, Viral , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H7N2 Subtype/genetics , Influenza in Birds/epidemiology , Zoonoses/epidemiology , Animals , Antigens, Viral/chemistry , Antigens, Viral/genetics , Antigens, Viral/metabolism , Binding Sites , Birds , Cat Diseases/transmission , Cat Diseases/virology , Cats , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Housing, Animal , Humans , Influenza A Virus, H7N2 Subtype/classification , Influenza A Virus, H7N2 Subtype/isolation & purification , Influenza in Birds/transmission , Influenza in Birds/virology , Models, Molecular , New York/epidemiology , Polysaccharides/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/metabolism , Veterinarians , Zoonoses/transmission , Zoonoses/virologyABSTRACT
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. Viruses isolated from the 41 specimens showed reduced or highly reduced inhibition by neuraminidase inhibition assay. iART may facilitate broader antiviral resistance testing.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral , Influenza A Virus, H1N1 Subtype/drug effects , Neuraminidase/antagonists & inhibitors , Oseltamivir/pharmacology , Antiviral Agents/therapeutic use , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human , Microbial Sensitivity Tests/methods , Neuraminidase/genetics , Neuraminidase/metabolism , Neuraminidase/therapeutic use , Oseltamivir/therapeutic useABSTRACT
Protein synthesis is a vital step in the successful replication of negative-strand RNA viruses. Protein synthesis is also a critical step in the development of a successful antiviral response from the host. This makes understanding the interplay between host and viral translation an important aspect of defining the virus/host interaction. For the negative-strand RNA viruses there are disparate mechanism of how viruses interact with the host protein synthesis apparatus, ranging from the complete takeover of all protein synthesis to the subtle insertion of viral mRNAs into an otherwise unchanged protein synthesis pattern. In this article, we discuss different ways to investigate protein synthesis in virus-infected cells, ranging from the use of metabolic labeling for the study of general translation changes to using fluorescence-coupled labeling techniques that allow the pinpointing of any subcellular localization of protein synthesis during virus replication. We also discuss methods for analyzing the translation initiation factors that are frequently modified in virus-infected cells.
Subject(s)
Host-Pathogen Interactions/genetics , Protein Biosynthesis/genetics , RNA Viruses/genetics , Viral Proteins/genetics , Animals , Genes, Reporter , Humans , RNA Viruses/metabolism , Viral Proteins/metabolismABSTRACT
Vesicular stomatitis virus (VSV) is a prototype nonsegmented, negative-sense virus used to examine viral functions of a broad family of viruses, including human pathogens. Here we demonstrate that S(2) VSV, an isolate with a small plaque phenotype compared to other Indiana strain viruses, has a transcription defect resulting in an altered pattern and rapid decline of transcription. The S(2) VSV transcription gradient is dominant over the wild-type transcription in a coinfection. This is the first characterization of an altered gradient of transcription not dependent on RNA template sequence or host response and may provide insight into new approaches to viral attenuation.
Subject(s)
Gene Expression Regulation, Viral , Transcription, Genetic , Vesiculovirus/physiology , Virus Replication , Animals , Cell Line , Cricetinae , RNA, Viral/biosynthesisABSTRACT
Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Orthopoxvirus/drug effects , Pyrimidinones/pharmacology , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Line , Humans , Molecular Structure , Orthopoxvirus/genetics , Orthopoxvirus/physiology , Poxviridae Infections/virology , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Small Molecule Libraries/chemical synthesis , Virus ReplicationABSTRACT
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 methods are laborious and require trained personnel. The influenza antiviral resistance test (iART), a prototype system developed by Becton, Dickinson and Company for research use only, offers a rapid and simple method to identify such viruses. This study investigated application of iART to influenza A viruses isolated from non-human hosts with a variety of NA subtypes (N1-N9).
ABSTRACT
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 assess antigenic relatedness between circulating viruses and vaccines. We developed a high content imaging-based neutralization test (HINT) to reduce antigenic mischaracterization resulting from virus adaptation to cell culture. Ferret reference antisera were raised using clinical specimens containing viruses representing recent vaccine strains. Analysis of viruses circulating during 2011-2018 showed that gain of an N158-linked glycosylation in HA was a molecular determinant of antigenic distancing between A/Hong Kong/4801/2014-like (clade 3C.2a) and A/Texas/50/2012-like viruses (clade 3C.1), while multiple evolutionary HA F193S substitution were linked to antigenic distancing from A/Switzerland/97152963/2013-like (clade 3C.3a) and further antigenic distancing from A/Texas/50/2012-like viruses. Additionally, a few viruses carrying HA T135K and/or I192T showed reduced neutralization by A/Hong Kong/4801/2014-like antiserum. Notably, this technique elucidated the antigenic characteristics of clinical specimens, enabling direct characterization of viruses produced in vivo, and eliminating in vitro culture, which rapidly alters the genotype/phenotype. HINT is a valuable new antigenic analysis tool for vaccine strain selection.
Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Hemagglutination Inhibition Tests/methods , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza, Human/immunology , Neutralization Tests/methods , Animals , Ferrets/immunology , Ferrets/virology , Glycosylation , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Immune Sera/immunology , Influenza A Virus, H3N2 Subtype/classification , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/physiology , Influenza Vaccines/immunology , Influenza, Human/diagnosis , Influenza, Human/virology , PhylogenyABSTRACT
Bangladesh has reported repeated outbreaks of highly pathogenic avian influenza (HPAI) A(H5) viruses in poultry since 2007. Because of the large number of live poultry markets (LPM) relative to the population density of poultry throughout the country, these markets can serve as sentinel sites for HPAI A(H5) detection. Through active LPM surveillance during June 2016-June 2017, HPAI A(H5N6) viruses along with 14 other subtypes of influenza A viruses were detected. The HPAI A(H5N6) viruses belonged to clade 2.3.4.4 and were likely introduced into Bangladesh around March 2016. Human infections with influenza clade 2.3.4.4 viruses in Bangladesh have not been identified, but the viruses had several molecular markers associated with potential human infection. Vigilant surveillance at the animal-human interface is essential to identify emerging avian influenza viruses with the potential to threaten public and animal health.
Subject(s)
Influenza A virus/isolation & purification , Influenza in Birds/virology , Poultry Diseases/virology , Animals , Bangladesh/epidemiology , Disease Outbreaks , Ducks , Evolution, Molecular , Geese/virology , Influenza A virus/classification , Influenza A virus/genetics , Influenza in Birds/epidemiology , Phylogeny , Poultry Diseases/epidemiologyABSTRACT
Influenza A viruses contain eight single-stranded, negative-sense RNA segments as viral genomes in the form of viral ribonucleoproteins (vRNPs). During genome replication in the nucleus, positive-sense complementary RNPs (cRNPs) are produced as replicative intermediates, which are not incorporated into progeny virions. To analyze the mechanism of selective vRNP incorporation into progeny virions, we quantified vRNPs and cRNPs in the nuclear and cytosolic fractions of infected cells, using a strand-specific qRT-PCR. Unexpectedly, we found that cRNPs were also exported to the cytoplasm. This export was chromosome region maintenance 1 (CRM1)-independent unlike that of vRNPs. Although both vRNPs and cRNPs were present in the cytosol, viral matrix (M1) protein, a key regulator for viral assembly, preferentially bound vRNPs over cRNPs. These results indicate that influenza A viruses selectively uptake cytosolic vRNPs through a specific interaction with M1 during viral assembly.
Subject(s)
Influenza A virus/genetics , RNA, Viral/genetics , Ribonucleoproteins/metabolism , Viral Matrix Proteins/metabolism , Virus Assembly/physiology , Virus Replication/physiology , A549 Cells , Active Transport, Cell Nucleus , Animals , Cell Line, Tumor , Cell Nucleus/virology , Cytoplasm/virology , Dogs , Genome, Viral/genetics , HEK293 Cells , Humans , Karyopherins/metabolism , Madin Darby Canine Kidney Cells , RNA, Viral/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Viral Matrix Proteins/genetics , Virus Assembly/genetics , Virus Replication/genetics , Exportin 1 ProteinABSTRACT
Paramyxoviruses replicate in the cytoplasm of infected cells and newly synthesized viral nucleocapsids (vRNPs) are transported to the plasma membrane to be incorporated into progeny virions. In this study, we analyzed the impact of the Rab11-mediated recycling pathway in Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) vRNP transport. We found that suppression of Rab11 expression caused vRNP aggregation in the cytoplasm and reduced progeny virion formation. Overexpression of constitutively active Rab11Q70L, but not dominant negative Rab11S25N co-localized with vRNP, showing that vRNP specifically recognizes the GTP-bound active form of Rab11. Moreover, Rab11Q70L co-localized with the dominant negative tails of all three subtypes of myosins, Va, Vb, and Vc, while SeV and hPIV1 vRNPs co-localized with only myosin Vb and Vc. These results highlight the critical role of Rab11 in vRNP trafficking, and suggest a specificity in the recycling endosomes parainfluenza viruses utilize for virus assembly.
Subject(s)
Myosin Type V/metabolism , Parainfluenza Virus 1, Human/metabolism , Sendai virus/metabolism , Virus Assembly/genetics , rab GTP-Binding Proteins/genetics , Animals , Cell Line , Endosomes/metabolism , HeLa Cells , Humans , Macaca mulatta , Nucleocapsid/metabolism , Parainfluenza Virus 1, Human/genetics , Paramyxoviridae Infections , Protein Transport/genetics , Protein Transport/physiology , RNA Interference , RNA, Small Interfering , Sendai virus/genetics , rab GTP-Binding Proteins/biosynthesis , rab4 GTP-Binding Proteins/metabolismABSTRACT
There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify chemical scaffolds for the development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses, suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.