Compounds derived from Humulus lupulus inhibit SARS-CoV-2 papain-like protease and virus replication.

BACKGROUND: Selected natural compounds exhibit very good antiviral properties. Especially, the medicinal plant Humulus lupulus (hop) contains several secondary plant metabolites some of which have previously shown antiviral activities. Among them, the prenylated chalcone xanthohumol (XN) demonstrated to be a potent inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). HYPOTHESIS/PURPOSE: Following the finding that xanthohumol (XN) is a potent inhibitor of SARS-CoV-2 Mpro, the effect of XN and its major derivatives isoxanthohumol (IXN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) from hops on SARS-CoV-2 papain-like protease (PLpro) were investigated. STUDY DESIGN: The modulatory effect of the hop compounds on PLpro were studied first in silico and then in vitro. In addition, the actual effect of hop compounds on the replication of SARS-CoV-2 in host cells was investigated. METHODS: In silico docking analysis was used to predict the binding affinity of hop compounds to the active site of PLpro. A recombinant PLpro was cloned, purified, characterized, and analyzed by small-angle X-ray scattering (SAXS), deISGylation assays, and kinetic analyses. Antiviral activity of hop compounds was assessed using the fluorescently labeled wildtype SARS-CoV-2 (icSARS-CoV-2-mNG) in Caco-2 host cells. RESULTS: Our in silico docking suggests that the purified hop compounds bind to the active site of SARS-CoV-2 PLpro blocking the access of its natural substrates. The hop-derived compounds inhibit SARS-CoV-2 PLpro with half maximal inhibitory concentration (IC50) values in the range of 59-162 µM. Furthermore, we demonstrate that XN and 6-PN, in particular, impede viral replication with IC50 values of 3.3 µM and 7.3 µM, respectively. CONCLUSION: In addition to the already known inhibition of Mpro by XN, our results show, for the first time, that hop-derived compounds target also SARS-CoV-2 PLpro which is a promising therapeutic target as it contributes to both viral replication and modulation of the immune system. These findings support the possibility to develop new hop-derived antiviral drugs targeting human coronaviruses.

Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspiramurdochii.

Brachyspira infections are significant causes of enterocolitis in pigs. In order to differentiate pathogenic species (Brachyspira (Br.) hyodysenteriae, Brachyspira pilosicoli) from less pathogenic or non-pathogenic species (Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii) in paraffin-embedded tissue samples a polymerase chain reaction (PCR) protocol allowing identification of Brachyspira at species level in archival material was developed. This approach was complemented by sequencing of the PCR amplification products. All seven cases presented with clinical and morphological Brachyspira-associated enterocolitis. Br. hyodysenteriae was not identified in any of the cases, while Br. pilosicoli was identified in a single case in conjunction with Br. murdochii. One case each was found positive for Br. innocens and Br. intermedia. Interestingly, the majority of cases presented as single or double infections with Br. murdochii. In some of the pigs other pathogens, like porcine circovirus-2 or Lawsonia intracellularis were present. These observations point at the possibility that under certain conditions even Brachyspira species of low pathogenicity can multiplicate extensively and lead to Brachyspira-associated enterocolitis.