Calcium dependent host innate immune resistance to influenza A virus infection

Influenza A virus infection is a major threat to public health, claiming over half a million lives globally each year. Strategies to combat influenza virus infection include vaccination and antiviral therapy. Influenza virus vaccine escape mutants, arising from mutations resulting in antigenic alterations of their surface glycoproteins so they are no longer recognised by the host immune system, can limit the effectiveness of vaccines. Secondly, antivirals that directly target the virus inevitably apply selection pressure on the virus and lead to drug resistance. The onset of resistance has been documented for the neuraminidase inhibitors, currently the only class of anti-influenza drugs recommended by the World Health Organisation. Targeting host factors that are required by the pathogen or enhancing host immune responses has been identified as an alternative to direct antiviral therapy. Host targeted treatment could be a viable alternative to overcome the serious problem of antiviral resistance. The evidence presented here indicates that activation of a specific host Ca2+ mobilisation pathway, store-operated calcium entry (SOCE), mediates a potent antiviral innate immune response that dramatically inhibits influenza virus replication in a variety of cell types. Non-cytotoxic doses of an SOCE agonist, a sesquiterpene lactone thapsigargin (TG), induced a sustained and broad spectrum innate immune state which was effective against the virus pre- and post-infection. TG priming induced the endoplasmic reticulum stress unfolded protein response and enhanced type I/III IFN associated response in the face of infection. In addition, TG priming reduced the expression of a range proviral host factors required for post-translational viral protein transport and modification. In summary, the strategic pharmacological or genetic activation of SOCE could underpin a new host-centric therapeutic approach capable of effectively inhibiting influenza A virus replication. TG was identified as a potential broad spectrum inhibitor of RNA virus replication capable of activating multiple host innate immune responses

Thapsigargin at non-cytotoxic levels induces a potent host antiviral response that blocks influenza a virus replication

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance

Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

The struggle to control the COVID-19 pandemic is made challenging by the emergence of virulent SARS-CoV-2 variants. To gain insight into their replication dynamics, emergent Alpha (A), Beta (B) and Delta (D) SARS-CoV-2 variants were assessed for their infection performance in single variant- and co-infections. The effectiveness of thapsigargin (TG), a recently discovered broad-spectrum antiviral, against these variants was also examined. Of the 3 viruses, the D variant exhibited the highest replication rate and was most able to spread to in-contact cells; its replication rate at 24h post-infection (hpi) based on progeny viral RNA production was over 4 times that of variant A and 9 times more than the B variant. In co-infections, the D variant boosted the replication of its co-infected partners at the expense of its own initial performance. Furthermore, co-infection with AD or AB combination conferred replication synergy where total progeny (RNA) output was greater than the sum of corresponding single-variant infections. All variants were highly sensitive to TG inhibition. A single pre-infection priming dose of TG effectively blocked all single-variant infections and every combination (AB, AD, BD variants) of co-infection at greater than 95% (relative to controls) at 72hpi. Likewise, TG was effective in inhibiting each variant in active preexisting infection. In conclusion, against the current backdrop of the dominant D variant that could be further complicated by co-infection synergy with new variants, the growing list of viruses susceptible to TG, a promising host-centric antiviral, now includes a spectrum of contemporary SARS-CoV-2 viruses

Extended 2D myotube culture recapitulates postnatal fibre type plasticity

Background: The traditional problems of performing skeletal muscle cell cultures derived from mammalian or avian species are limited myotube differentiation, and transient myotube persistence which greatly restricts the ability of myotubes to undergo phenotypic maturation. We report here on a major technical breakthrough in the establishment of a simple and effective method of extended porcine myotube cultures (beyond 50 days) in two-dimension (2D) that recapitulates key features of postnatal fibre types. Results: Primary porcine muscle satellite cells (myoblasts) were isolated from the longissimus dorsi of 4 to 6 weeks old pigs for 2D cultures to optimise myotube formation, improve surface adherence and characterise myotube maturation. Over 95 % of isolated cells were myoblasts as evidenced by the expression of Pax3 and Pax7. Our relatively simple approach, based on modifications of existing surface coating reagents (Maxgel), and of proliferation and differentiation (Ultroser G) media, typically achieved by 5 days of differentiation fusion index of around 80 % manifested in an abundance of discrete myosin heavy chain (MyHC) slow and fast myotubes. There was little deterioration in myotube viability over 50 days, and the efficiency of myotube formation was maintained over seven myoblast passages. Regular spontaneous contractions of myotubes were frequently observed throughout culture. Myotubes in extended cultures were able to undergo phenotypic adaptation in response to different culture media, including the adoption of a dominant postnatal phenotype of fast-glycolytic MyHC 2x and 2b expression by about day 20 of differentiation. Furthermore, fast-glycolytic myotubes coincided with enhanced expression of the putative porcine long intergenic non-coding RNA (linc-MYH), which has recently been shown to be a key coordinator of MyHC 2b expression in vivo. Conclusions: Our revised culture protocol allows the efficient differentiation and fusion of porcine myoblasts into myotubes and their prolonged adherence to the culture surface. Furthermore, we are able to recapitulate in 2D the maturation process of myotubes to resemble postnatal fibre types which represent a major technical advance in opening access to the in vitro study of coordinated postnatal muscle gene expression

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to in-clude antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of an-tivirals in use or development for any disease bears testament to the challenges of antiviral de-velopment. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic / endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate in-fections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus

Calcium dependent host innate immune resistance to influenza A virus infection

Influenza A virus infection is a major threat to public health, claiming over half a million lives globally each year. Strategies to combat influenza virus infection include vaccination and antiviral therapy. Influenza virus vaccine escape mutants, arising from mutations resulting in antigenic alterations of their surface glycoproteins so they are no longer recognised by the host immune system, can limit the effectiveness of vaccines. Secondly, antivirals that directly target the virus inevitably apply selection pressure on the virus and lead to drug resistance. The onset of resistance has been documented for the neuraminidase inhibitors, currently the only class of anti-influenza drugs recommended by the World Health Organisation. Targeting host factors that are required by the pathogen or enhancing host immune responses has been identified as an alternative to direct antiviral therapy. Host targeted treatment could be a viable alternative to overcome the serious problem of antiviral resistance. The evidence presented here indicates that activation of a specific host Ca2+ mobilisation pathway, store-operated calcium entry (SOCE), mediates a potent antiviral innate immune response that dramatically inhibits influenza virus replication in a variety of cell types. Non-cytotoxic doses of an SOCE agonist, a sesquiterpene lactone thapsigargin (TG), induced a sustained and broad spectrum innate immune state which was effective against the virus pre- and post-infection. TG priming induced the endoplasmic reticulum stress unfolded protein response and enhanced type I/III IFN associated response in the face of infection. In addition, TG priming reduced the expression of a range proviral host factors required for post-translational viral protein transport and modification. In summary, the strategic pharmacological or genetic activation of SOCE could underpin a new host-centric therapeutic approach capable of effectively inhibiting influenza A virus replication. TG was identified as a potential broad spectrum inhibitor of RNA virus replication capable of activating multiple host innate immune responses