Growth activation of influenza virus by trypsin and effect of T-705 (favipiravir) on trypsin-optimized growth condition

Abstract

Influenza virus is activated by proteolytic cleavage of hemagglutinin by trypsin. After determining the optimal trypsin concentration, intracellular and extracellular influenza A/PR/8/34 (H1N1) and A/ Victoria/361/2011 (H3N2) virus productions were compared in cultures treated with T-705 (favipiravir) and GS 4071 (an active form of oseltamivir). Although both drugs efficiently inhibited extracellular viral RNA release in a dose-dependent manner, T-705 inhibited it to the level of the inoculum without trypsin treatment, while GS 4071 inhibited it to a final level 10 times higher than that without trypsin. T-705 inhibited intracellular viral RNA production to the level of input virus in both trypsin-treated and untreated cells. In contrast, GS 4071 dosedependently inhibited intracellular viral RNA production in cells treated with trypsin but allowed viral RNA synthesis. The level of maximum inhibition by GS 4071was 10 times higher than that of cells without trypsin and 1,000 times greater than the inoculum titer in cells without trypsin. T-705 inhibited both intracellular and extracellular virus production 1,000 and 10 times more strongly, respectively, than GS 4071. T-705 has powerful anti-influenza activity in the absence of trypsin and even in the trypsin-optimized growth condition, suggesting the therapeutic advantage in treatment of influenza complicated with bacterial pneumonia.

Does Trypsin Oral Spray (Viruprotect®/ColdZyme®) Protect against COVID-19 and Common Colds or Induce Mutation? Caveats in Medical Device Regulations in the European Union

“…Table S1 summarizes detailed information on the assays and methods applied, and the key findings of the studies [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Virus resistance to trypsin, virus survival and enhanced or sustained cell invasion and syncytia formation with trypsin were also reported in host cell assays with rhinovirus, RSV and influenza [ 40 , 41 , 42 , 43 , 44 , 45 ].…”

“…Yet, no information in clinical trial protocols appear to have addressed the potential health hazards that were identified from known effects of trypsin/glycerin: these include (1) increased infectivity of respiratory viruses on trypsin exposure (as seen with trypsin in host cells in absence of trypsin inhibition), not only for SARS-CoV-2 ( Table S1 , [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]) but also for influenza and the Respiratory Syncytial Virus (RSV) [ 40 , 42 , 43 ], (2) induction of irritation and constriction of airways by inhaling glycerin/trypsin [ 61 , 62 ], and (3) epithelial damage and/or digestion of the protective mucin layer by trypsin possible at the level of labial, pharyngeal, oesophageal and gastric mucosa [ 63 , 64 , 65 ], (4) so trypsin/glycerin possibly contributing to hoarseness, herpes labialis eruption, gastro-esophageal reflux disease, and gastric or respiratory problems, such as occasionally reported in clinical studies or rather assessed by the manufacturer as a CC symptom [ 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 ]. Leaflets mention a caution for allergy, while it is asked not to breath during spraying, “because aspiration can cause transient-asthma-like symptoms, such as cough and hoarseness” [ 7 ].…”

“…Additionally, for the other coronaviruses listed in the Table S1 , none of the independent studies showed a potential benefit of exogenous trypsin added to cell cultures for inhibiting viral invasion. Moreover, our additional searches on other respiratory viruses supported that—opposite to the desired benefit—trypsin is typically added to induce cleavage and activation of viral proteins, and/or to promote viral growth in cells, such as for RSV [ 40 ], influenza virus [ 42 , 43 ] and human metapneumovirus [ 83 ]. Furthermore, conformational dynamic studies of the SARS-CoV-2 spikes confirmed a role of trypsin in activating the spikes, when opening in response to human Angiotensin-converting enzyme 2 (ACE2), its impact allowing easier human ACE2-bound spike conformation [ 84 ].…”

Advances in the development of antivirals for rotavirus infection

“…It is widely believed that trypsin-like proteases promote rotavirus replication in vivo and in vitro, and protease hydrolysis causes PDCoV (73), TuMV (74), influenza A and B viruses (75), reovirus, and rotavirus (76). The small intestine is the main site of rotavirus replication where there are high levels of protease activity.…”