Assessing replication, transmission and fitness of influenza viruses with reduced susceptibility to neuraminidase inhibitors

© 2019 Rubaiyea FarrukeeInfluenza viruses can cause severe respiratory disease and are responsible for significant morbidity and mortality worldwide. Antivirals are valuable for treatment of influenza infections and neuraminidase inhibitors (NAIs) (oseltamivir, peramivir, laninamivir and zanamivir), are the most commonly available influenza antivirals. NAIs work by blocking the enzymatic activity of the viral neuraminidase (NA) protein, however, viral susceptibility to NAIs can be reduced due to amino acid substitutions that arise in the NA protein. The emergence of variants with reduced NAI susceptibility is a major public health concern. Therefore, the studies described in this thesis aimed to understand the biology of variant influenza viruses with amino acid substitutions in the viral NA protein that reduce sensitivity to NAI in vitro, focussing on their impact on drug effectiveness in vivo, viral fitness, and the risk of emergence and spread. First, we aimed to understand how amino acid substitutions that increase IC50 values (in 50% inhibitory capacity of NAIs), impact drug effectiveness in vivo. Utilising a ferret model, we were able to demonstrate a direct correlation between increased IC50 and reduced effectiveness of oseltamivir, the most widely used NAI, in ferrets. The findings from these experiments highlights the need for revising current guidelines pertaining to influenza B variants, and the risk posed by variants with the commonly reported H273Y or D197N NA (B numbering) substitutions. These substitutions were further characterized in recent viral backgrounds and their impact on NA enzyme function, viral replication and transmission was determined. These experiments demonstrated that both substitutions reduced viral fitness, but the H273Y substitution reduced fitness more than the D197N substitution. We also aimed to understand the risk of variants with reduced NAI sensitivity emerging in A(H7N9) viruses, and selected for drug escape variants by serial passaging reassorted N9 viruses in increasing NAI pressure. These experiments revealed that oseltamivir, peramivir and laninamivir selected for variants with reduced NAI susceptibility, but zanamivir did not. Further analysis on the impact of different amino acid substitutions on N9 NAs derived from different viral backgrounds revealed that with each substitution there was a greater loss of enzyme function in the NA from a Yangtze-River lineage virus than from a Pearl-River Delta lineage virus. The same amino acid substitution can have a different fitness impact in different backgrounds. This was observed with the H275Y substitution (N1 numbering), which was more readily accommodated in 2007 A/Brisbane-like seasonal A(H1N1) viruses compared to earlier A(H1N1) viruses due to permissive substitutions, and therefore became widespread in the human population. In order to predict the risk of H275Y becoming widespread in currently circulating A(H1N1)pdm09 viruses, we established a combination of computational and experimental protocols to predict potentially permissive substitutions in a recent A(H1N1)pdm09 virus, and were able to propose a number of candidate substitutions that will need further evaluation. Overall these studies provided important insights into variant influenza viruses with amino acid substitutions in the viral NA protein that reduce sensitivity to NAI in vitro, particularly in regards to their risk of reducing drug effectiveness and becoming widespread. Furthermore, we have established a number of experimental and computational approaches which can be useful for characterizing fitness of variants with reduced susceptibility to novel antivirals entering the market

A rapid pyrosequencing assay for the molecular detection of influenza viruses with reduced baloxavir susceptibility due to PA/I38X amino acid substitutions

Baloxavir marboxil is a novel endonuclease inhibitor licensed for treatment of otherwise healthy or high-risk individuals infected with influenza. Viruses with reduced baloxavir susceptibility due to amino acid substitutions at residue 38 of the PA have been detected in some individuals following treatment. Here, we describe a genotypic pyrosequencing method that can be used to rapidly screen circulating influenza A and B viruses for substitutions in the PA/I38 codon and to quantify mixed viral populations. This method is suitable for surveillance of baloxavir susceptibility and to analyse samples from hospitalised patients undergoing baloxavir treatment to aid in clinical decision making

Host Cell Restriction Factors of Paramyxoviruses and Pneumoviruses

The paramyxo- and pneumovirus family includes a wide range of viruses that can cause respiratory and/or systemic infections in humans and animals. The significant disease burden of these viruses is further exacerbated by the limited therapeutics that are currently available. Host cellular proteins that can antagonize or limit virus replication are therefore a promising area of research to identify candidate molecules with the potential for host-targeted therapies. Host proteins known as host cell restriction factors are constitutively expressed and/or induced in response to virus infection and include proteins from interferon-stimulated genes (ISGs). Many ISG proteins have been identified but relatively few have been characterized in detail and most studies have focused on studying their antiviral activities against particular viruses, such as influenza A viruses and human immunodeficiency virus (HIV)-1. This review summarizes current literature regarding host cell restriction factors against paramyxo- and pneumoviruses, on which there is more limited data. Alongside discussion of known restriction factors, this review also considers viral countermeasures in overcoming host restriction, the strengths and limitations in different experimental approaches in studies reported to date, and the challenges in reconciling differences between in vitro and in vivo data. Furthermore, this review provides an outlook regarding the landscape of emerging technologies and tools available to study host cell restriction factors, as well as the suitability of these proteins as targets for broad-spectrum antiviral therapeutics

Characterization of Influenza B Virus Variants with Reduced Neuraminidase Inhibitor Susceptibility

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution

Isoforms of Human MARCH1 Differ in Ability to Restrict Influenza A Viruses Due to Differences in Their N Terminal Cytoplasmic Domain

MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known regarding virus inhibition by MARCH1. Isoforms 1 and 2 of MARCH1 are very similar in overall structure but show major differences in their N-terminal cytoplasmic domain (N-CT). Herein, we used a doxycycline-inducible overexpression system to demonstrate that MARCH1.1 reduces titres of influenza A virus (IAV) released from infected cells whereas MARCH1.2 does not. The deletion of the entire N-CT of MARCH1.2 restored its ability to restrict IAV infectivity and sequential deletions mapped the restoration of IAV inhibition to delete the 16 N-terminal residues within the N-CT of MARCH1.2. While only MARCH1.1 mediated anti-IAV activity, qPCR demonstrated the preferential expression of MARCH1.2 over MARCH1.1 mRNA in unstimulated human peripheral blood mononuclear cells and also in monocyte-derived macrophages. Together, these studies describe the differential ability of MARCH1 isoforms to restrict IAV infectivity for the first time. Moreover, as published immunological, virological and biochemical studies examining the ability of MARCH1 to target particular ligands generally use only one of the two isoforms, these findings have broader implications for our understanding of how MARCH1 isoforms might differ in their ability to modulate particular host and/or viral proteins

TRIM16 Overexpression in HEK293T Cells Results in Cell Line-Specific Antiviral Activity

Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity

Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility

Objectives: The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidase mutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like). Methods: We characterized the effect of 16 amino acid substitutions across five framework residues and four monomeric interface residues on the susceptibility to four different NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Results: Framework residue mutations E117A and E117G conferred highly reduced inhibition to three of the four NAIs, but substantially reduced neuraminidase activity, whereas other framework mutations retained a greater level of NA activity. Mutations E105K, P139S and G140R of the monomeric interface were also found to cause highly reduced inhibition, but, interestingly, their effect was substantially greater in a B/Victoria-like neuraminidase than in a B/Yamagata-like neuraminidase, with some susceptibility values being up to 1000-fold different between lineages. Conclusions: The frequency and the effect of key neuraminidase mutations on neuraminidase activity and NAI susceptibility can differ substantially between the two influenza B lineages. Therefore, future surveillance, analysis and interpretation of influenza B virus NAI susceptibility should consider the B lineage of the neuraminidase in the same manner as already occurs for different influenza A neuraminidase subtypes.Rubaiyea Farrukee, Sook-Kwan Leang, Jeff Butler, Raphael T.C. Lee, Sebastian Maurer-Stroh, Danielle Tilmanis, Sheena Sullivan, Jennifer Mosse, Ian G. Barr and Aeron C. Hur

Retinoic Acid–Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection

Abstract Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid–inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an “antiviral state.” In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis

DC-SIGN and L-SIGN are attachment factors that promote infection of target cells by human metapneumovirus in the presence or absence of cellular glycosaminoglycans

It is well established that glycosaminoglycans (GAGs) function as attachment factors for human metapneumovirus (HMPV), concentrating\ud virions at the cell surface to promote interaction with other receptors for virus entry and infection. There is increasing\ud evidence to suggest that multiple receptors may exhibit the capacity to promote infectious entry of HMPV into host cells;\ud however, definitive identification of specific transmembrane receptors for HMPV attachment and entry is complicated by the\ud widespread expression of cell surface GAGs. pgsA745 Chinese hamster ovary (CHO) cells are deficient in the expression of cell\ud surface GAGs and resistant to HMPV infection. Here, we demonstrate that the expression of the Ca²⁺-dependent C-type lectin receptor (CLR) DC-SIGN (CD209L) or L-SIGN (CD209L) rendered pgsA745 cells permissive to HMPV infection.\ud Unlike infection of parental CHO cells, HMPV infection of pgsA745 cells expressing DC-SIGN or L-SIGN was dynamin dependent\ud and inhibited by mannan but not by pretreatment with bacterial heparinase. Parental CHO cells expressing DC-SIGN/L-SIGN also\ud showed enhanced susceptibility to dynamin-dependent HMPV infection, confirming that CLRs can promote HMPV infection in the\ud presence or absence of GAGs. Comparison of pgsA745 cells expressing wild-type and endocytosis-defective mutants of DC-SIGN/L-SIGN\ud indicated that the endocytic function of CLRs was not essential but could contribute to HMPV infection of GAG-deficient cells.\ud Together, these studies confirm a role for CLRs as attachment factors and entry receptors for HMPV infection. Moreover, they\ud define an experimental system that can be exploited to identify transmembrane receptors and entry pathways where permissivity\ud to HMPV infection can be rescued following the expression of a single cell surface receptor

Utilising animal models to evaluate oseltamivir efficacy against influenza A and B viruses with reduced in vitro susceptibility.

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility