Novel isoforms of influenza virus PA-X and PB1-F2 indicated by automatic annotation.

The influenza A virus genome contains 8 gene segments encoding 10 commonly recognized proteins. Additional protein products have been identified, including PB1-F2 and PA-X. We report the in-silico identification of novel isoforms of PB1-F2 and PA-X in influenza virus genomes sequenced from avian samples. The isoform observed in PA-X includes a mutated stop codon that should extend the protein product by 8 amino acids. The isoform observed in PB1-F2 includes two nonsense mutations that should truncate the N-terminal region of the protein product and remove the entire mitochondrial targeting domain. Both isoforms were uncovered during automatic annotation of CEIRS sequence data. Nominally termed PA-X8 and PB1-F2-Cterm, both predicted isoforms were subsequently found in other annotated influenza genomes previously deposited in GenBank. Both isoforms were noticed due to discrepant annotations output by two annotation engines, indicating a benefit of incorporating multiple algorithms during gene annotation.

Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance-Associated Substitutions in Ferrets.

Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)-generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg-wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6-4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A.

Competitive fitness of influenza B viruses with neuraminidase inhibitor-resistant substitutions in a coinfection model of the human airway epithelium.

UNLABELLED: Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. IMPORTANCE: Influenza B viruses are important human respiratory pathogens contributing to a significant portion of seasonal influenza virus infections worldwide. The development of resistance to a single class of available antivirals, the neuraminidase (NA) inhibitors (NAIs), is a public health concern. Amino acid substitutions in the NA glycoprotein of influenza B virus not only can confer antiviral resistance but also can alter viral fitness. Here we used normal human bronchial epithelial (NHBE) cells, a model of the human upper respiratory tract, to examine the replicative capacities and fitness of NAI-resistant influenza B viruses. We show that virus with an E119A NA substitution can replicate efficiently in NHBE cells in the presence of oseltamivir or zanamivir and that virus with the H274Y NA substitution has a relative fitness greater than that of the wild-type NAI-susceptible virus. This study is the first to use NHBE cells to determine the fitness of NAI-resistant influenza B viruses.

Fitness costs for Influenza B viruses carrying neuraminidase inhibitor-resistant substitutions: underscoring the importance of E119A and H274Y.

Influenza B viruses cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated pediatric mortality. Neuraminidase (NA) inhibitors (NAIs) are the only available therapy for patients infected with influenza B viruses, and the potential emergence of NAI-resistant viruses is a public health concern. The NA substitutions located within the enzyme active site could not only reduce NAI susceptibility of influenza B virus but also affect virus fitness. In this study, we investigated the effect of single NA substitutions on the fitness of influenza B/Yamanashi/166/1998 viruses (Yamagata lineage). We generated recombinant viruses containing either wild-type (WT) NA or NA with a substitution in the catalytic (R371K) or framework (E119A, D198E, D198Y, I222T, H274Y, and N294S) residues. We assessed NAI susceptibility, NA biochemical properties, NA protein expression, and virus replication in vitro and in differentiated normal human bronchial epithelial (NHBE) cells. Our results showed that four NA substitutions (D198E, I222T, H274Y, and N294S) conferred reduced inhibition by oseltamivir and three (E119A, D198Y, and R371K) conferred highly reduced inhibition by oseltamivir, zanamivir, and peramivir. All NA substitutions, except for D198Y and R371K, were genetically stable after seven passages in MDCK cells. Cell surface NA protein expression was significantly increased by H274Y and N294S substitutions. Viruses with the E119A, I222T, H274Y, or N294S substitution were not attenuated in replication efficiency in vitro or in NHBE cells. Overall, viruses with the E119A or H274Y NA substitution possess fitness comparable to NAI-susceptible virus, and the acquisition of these substitutions by influenza B viruses should be closely monitored.

The neuraminidase inhibitor oseltamivir is effective against A/Anhui/1/2013 (H7N9) influenza virus in a mouse model of acute respiratory distress syndrome.

BACKGROUND: High mortality and uncertainty about the effectiveness of neuraminidase inhibitors (NAIs) in humans infected with influenza A(H7N9) viruses are public health concerns. METHODS: Susceptibility of N9 viruses to NAIs was determined in a fluorescence-based assay. The NAI oseltamivir (5, 20, or 80 mg/kg/day) was administered to BALB/c mice twice daily starting 24, 48, or 72 hours after A/Anhui/1/2013 (H7N9) virus challenge. RESULTS: All 12 avian N9 and 3 human H7N9 influenza viruses tested were susceptible to NAIs. Without prior adaptation, A/Anhui/1/2013 (H7N9) caused lethal infection in mice that was restricted to the respiratory tract and resulted in pulmonary edema and acute lung injury with hyaline membrane formation, leading to decreased oxygenation, all characteristics of human acute respiratory distress syndrome. Oseltamivir at 20 and 80 mg/kg protected 80% and 88% of mice when initiated after 24 hours, and the efficacy decreased to 70% and 60%, respectively, when treatment was delayed by 48 hours. Emergence of oseltamivir-resistant variants was not detected. CONCLUSIONS: H7N9 viruses are comparable to currently circulating influenza A viruses in susceptibility to NAIs. Based on these animal studies, early treatment is associated with improved outcomes.

Neuraminidase inhibitors for influenza B virus infection: efficacy and resistance.

Many aspects of the biology and epidemiology of influenza B viruses are far less studied than for influenza A viruses, and one of these aspects is efficacy and resistance to the clinically available antiviral drugs, the neuraminidase (NA) inhibitors (NAIs). Acute respiratory infections are one of the leading causes of death in children and adults, and influenza is among the few respiratory infections that can be prevented and treated by vaccination and antiviral treatment. Recent data has suggested that influenza B virus infections are of specific concern to pediatric patients because of the increased risk of severe disease. Treatment of influenza B is a challenging task for the following reasons: This review presents current knowledge of the efficacy of NAIs for influenza B virus and antiviral resistance in clinical, surveillance, and experimental studies.

Role for subgenomic mRNA in host translation inhibition during Sindbis virus infection of mammalian cells.

Sindbis virus subgenomic mRNA is efficiently translated in infected vertebrate cells whereas host translation is shut-off. Deletions in the 5'UTR of the subgenomic mRNA were made to investigate its role in viral gene expression. Deletion of nucleotides 1-10 and 11-20 caused a small plaque phenotype, reduced levels of subgenomic mRNA and structural proteins, and increased expression of nonstructural proteins. Whereas deletion 1-10 virus inhibited cellular protein synthesis, deletion 11-20 did so inefficiently. A large plaque revertant of deletion 11-20, possessing a duplication of the subgenomic promoter region, produced subgenomic mRNA at WT levels and restored inhibition of host protein synthesis. Further analysis of the mutant and revertant 5'UTR sequences showed the ability to shut-off host cell translation correlated with the efficiency of translation of subgenomic mRNA. We propose that the translational efficiency and quantity of the subgenomic mRNA play a role in inhibition of host cell translation.

Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA.

Alphaviruses are a group of arthropod-borne human and animal pathogens that can cause epidemics of significant public health and economic consequence. Alphavirus RNA synthesis requires four virally encoded nonstructural proteins and probably a number of cellular proteins. Using comparative two-dimensional electrophoresis we were able to identify proteins enriched in cytoplasmic membrane fractions containing viral RNA synthetic complexes following infection with Sindbis virus. Our studies demonstrated the following: (i) the host protein hnRNP K is enriched in cytoplasmic membrane fractions following Sindbis virus infection, (ii) viral nonstructural proteins co-immunoprecipitate with hnRNP K, (iii) nsP2 and hnRNP K co-localize in the cytoplasm of Sindbis virus infected cells, (iv) Sindbis virus subgenomic mRNA, but not genomic RNA co-immunoprecipitates with hnRNP K, (v) viral RNA does not appear to be required for the interaction of hnRNP K with the nonstructural proteins. Potential functions of hnRNP K during virus replication are discussed.