Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Abstract

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

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“…On the contrary, the 19 independent studies that we reviewed ( Table S1 ) consistently showed that exogenous trypsin added to host cells in presence of SARS-CoV-1 ( n = 3), SARS-CoV-2 ( n = 10) and other coronaviruses ( n = 7) does not annihilate nor stop, but facilitates priming of the spike and/or cell entry, and enhances virus infectivity, cell fusion and syncytia formation in vitro [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. The effects were obtained in the ng to µg trypsin/mL range, so likely at lower (and thus relevant) concentrations than obtained with the 1.2 mL volume used in the manufacturer’s virucidal suspension test.…”

“…The effects were obtained in the ng to µg trypsin/mL range, so likely at lower (and thus relevant) concentrations than obtained with the 1.2 mL volume used in the manufacturer’s virucidal suspension test. 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 ].…”

“…The literature analysis in Table S1 also revealed several studies indicating that serial passage of virus in cell culture in the presence of trypsin induced mutations, identified in spike (S) and other proteins of SARS-CoV-2 and other coronaviruses [ 28 , 31 ]. Mutants were described for their impact in presence versus absence of trypsin.…”

“…The latter hits were retained if referring to SARS-CoV-1 or other coronaviruses, while excluded if referring to absence of trypsin ( n = 2), trypsin-like activity or other proteases ( n = 3), review-type of publication ( n = 3), protein analysis ( n = 2), trypsin-1 gene variants or vaccine ( n = 2), alpha-1-anti-trypsin ( n = 1) or lactoferrin preparation ( n = 1), resulting in 11 manufacturer-independent sources on trypsin. Nine more sources on the use of exogenous trypsin related to SARS-CoV(2) were identified during the literature study via citations in these articles and were tabulated as well ( Supplementary Table S1 ) [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Information on mutants was generated from this set of publications.…”