Naïve Human Macrophages Are Refractory to SARS-CoV-2 Infection and Exhibit a Modest Inflammatory Response Early in Infection.

Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-α and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 exposed MDM with notable absence of IFN-β induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon exposure to virus. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages

The mechanism of resistance to favipiravir in influenza.

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance

Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase

Avian influenza A viruses often do not propagate efficiently in mammalian cells. The viral polymerase protein PB2 is important for this host restriction, with amino-acid polymorphisms at residue 627 and other positions acting as ‘signatures’ of avian- or human-adapted viruses. Restriction is hypothesized to result from differential interactions (either positive or inhibitory) with unidentified cellular factors. We applied fluorescence recovery after photobleaching (FRAP) to investigate the mobility of the viral polymerase in the cell nucleus using A/PR/8/34 and A/Turkey/England/50-92/91 as model strains. As expected, transcriptional activity of a polymerase with the avian PB2 protein was strongly dependent on the identity of residue 627 in human but not avian cells, and this correlated with significantly slower diffusion of the inactive polymerase in human but not avian nuclei. In contrast, the activity and mobility of the PR8 polymerase was affected much less by residue 627. Sequence comparison followed by mutagenic analyses identified residues at known host-range-specific positions 271, 588 and 701 as well as a novel determinant at position 636 as contributors to host-specific activity of both PR8 and Turkey PB2 proteins. Furthermore, the correlation between poor transcriptional activity and slow diffusional mobility was maintained. However, activity did not obligatorily correlate with predicted surface charge of the 627 domain. Overall, our data support the hypothesis of a host nuclear factor that interacts with the viral polymerase and modulates its activity. While we cannot distinguish between positive and inhibitory effects, the data have implications for how such factors might operate

Low dose influenza virus challenge in the ferret leads to increased virus shedding and greater sensitivity to oseltamivir

Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 106 plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 102 pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 104 pfu gave an infection that was intermediate between those of the 106 pfu and 102 pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (106 pfu) and low (102 pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines

Amino acid substitutions in the H5N1 avian influenza haemagglutinin alter pH of fusion and receptor binding to promote a highly pathogenic phenotype in chickens

Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo. A third novel substitution in the fusion peptide, HA2G4R, enabled the HP phenotype. HA2G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS

Swine ANP32A supports avian influenza virus polymerase

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as 'mixing vessels', being susceptible to both avian and human origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports activity of avian origin influenza virus polymerases, and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced pro-viral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the 'mixing vessel' trait of swine and further our understanding of the evolution and ecology of viruses in this host.Importance Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus 'mixing vessels'. In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this through binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as 'mixing vessels'

An influenza A virus can evolve to use human ANP32E through altering polymerase dimerization

Human ANP32A and ANP32B are essential but redundant host factors for influenza virus genome replication. While most influenza viruses cannot replicate in edited human cells lacking both ANP32A and ANP32B, some strains exhibit limited growth. Here, we experimentally evolve such an influenza A virus in these edited cells and unexpectedly, after 2 passages, we observe robust viral growth. We find two mutations in different subunits of the influenza polymerase that enable the mutant virus to use a novel host factor, ANP32E, an alternative family member, which is unable to support the wild type polymerase. Both mutations reside in the symmetric dimer interface between two polymerase complexes and reduce polymerase dimerization. These mutations have previously been identified as adapting influenza viruses to mice. Indeed, the evolved virus gains the ability to use suboptimal mouse ANP32 proteins and becomes more virulent in mice. We identify further mutations in the symmetric dimer interface which we predict allow influenza to adapt to use suboptimal ANP32 proteins through a similar mechanism. Overall, our results suggest a balance between asymmetric and symmetric dimers of influenza virus polymerase that is influenced by the interaction between polymerase and ANP32 host proteins

A common TMPRSS2 variant has a protective effect against severe COVID-19

Background : The human protein transmembrane protease serine type 2 (TMPRSS2) plays a key role in SARS-CoV-2 infection, as it is required to activate the virus’ spike protein, facilitating entry into target cells. We hypothesized that naturally-occurring TMPRSS2 human genetic variants affecting the structure and function of the TMPRSS2 protein may modulate the severity of SARS-CoV-2 infection. Methods : We focused on the only common TMPRSS2 non-synonymous variant predicted to be damaging (rs12329760 C>T, p.V160M), which has a minor allele frequency ranging from from 0.14 in Ashkenazi Jewish to 0.38 in East Asians. We analysed the association between the rs12329760 and COVID-19 severity in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units recruited as part of the GenOMICC (Genetics Of Mortality In Critical Care) study. Logistic regression analyses were adjusted for sex, age and deprivation index. For in vitro studies, HEK293 cells were co-transfected with ACE2 and either TMPRSS2 wild type or mutant (TMPRSS2V160M). A SARS-CoV-2 pseudovirus entry assay was used to investigate the ability of TMPRSS2V160M to promote viral entry. Results : We show that the T allele of rs12329760 is associated with a reduced likelihood of developing severe COVID-19 (OR 0.87, 95%CI:0.79-0.97, p=0.01). This association was stronger in homozygous individuals when compared to the general population (OR 0.65, 95%CI:0.50-0.84, p=1.3 × 10−3). We demonstrate in vitro that this variant, which causes the amino acid substitution valine to methionine, affects the catalytic activity of TMPRSS2 and is less able to support SARS-CoV-2 spike-mediated entry into cells. Conclusion : TMPRSS2 rs12329760 is a common variant associated with a significantly decreased risk of severe COVID-19. Further studies are needed to assess the expression of TMPRSS2 across different age groups. Moreover, our results identify TMPRSS2 as a promising drug target, with a potential role for camostat mesilate, a drug approved for the treatment of chronic pancreatitis and postoperative reflux esophagitis, in the treatment of COVID-19. Clinical trials are needed to confirm this

The antiviral potential of the antiandrogen enzalutamide and the viral‐androgen signaling interplay in seasonal coronaviruses

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions