Identification and characterization of the activated defence response in the commercially important Agarophyte, Gracilaria Gracilis, following exposure to disease elicitors

To our knowledge, this study represents the first analysis of gene expression using cDNA microarrays in the red macroalga G. gracilis. Western hybridization analysis was used to establish whether the observed changes in gene expression following exposure to disease elicitors positively correlated to changes at the protein level

Evaluation of a human mucosal tissue explant model for SARS-CoV-2 replication.

With the onset of COVID-19, the development of ex vivo laboratory models became an urgent priority to study host-pathogen interactions in response to the pandemic. In this study, we aimed to establish an ex vivo mucosal tissue explant challenge model for studying SARS-CoV-2 infection and replication. Nasal or oral tissue samples were collected from eligible participants and explants generated from the tissue were infected with various SARS-CoV-2 strains, including IC19 (lineage B.1.13), Beta (lineage B.1.351) and Delta (lineage B.1.617.2). A qRT-PCR assay used to measure viral replication in the tissue explants over a 15-day period, demonstrated no replication for any viral strains tested. Based on this, the ex vivo challenge protocol was modified by reducing the viral infection time and duration of sampling. Despite these changes, viral infectivity of the nasal and oral mucosa was not improved. Since 67% of the enrolled participants were already vaccinated against SARS-CoV-2, it is possible that neutralizing antibodies in explant tissue may have prevented the establishment of infection. However, we were unable to optimize plaque assays aimed at titrating the virus in supernatants from both infected and uninfected tissue, due to limited volume of culture supernatant available at the various collection time points. Currently, the reasons for the inability of these mucosal tissue samples to support replication of SARS-CoV-2 ex vivo remains unclear and requires further investigation

Optimisation of E484K multiplex qPCR assays for Beta variant calling.

(A) Schematic of genome organization of SARS-CoV-2 coronavirus showing primer pairs and MGB probes targeting the E484K SNP within the Spike gene. Enlarged region corresponds to the E484K SNP (blue line within brown box) with flanking primers (S-F1 and S-R2 depicted by green boxes) and probes (brown box) for multiplex assay (cognate sequence for Wuhan-specific probe = 484-Wuhan fluorescently labelled with Cy5; cognate sequence for Beta-specific probe = 484-Beta fluorescently labelled with Rox). (B) Amplification plot and melt curve analysis (SYBR green chemistry) of S-F1R1 primer pair to confirm primer specificity using cDNA derived from Wuhan-Hu-1. A single peak in the melt-curve analysis is indicative of primer specificity. (C) Amplification curves of fluorescence intensity versus cycle threshold (CT) with E484K primer-probe sets of multiplex qPCR. The Wuhan-Hu-1 (Cy5) and Beta (Rox) MGB probes targeting the variant-defining SNPs were combined in a single reaction and tested using cDNA derived from either of the two strains. Both MGB probes were highly specific and only amplified cDNA containing its cognate SNP. The no template control reactions showed no amplification and were void of any contaminating DNA. All qPCR reactions were set up in duplicate. (TIF)</p

<i>Ex vivo</i> challenge of oral and nasal tissue from study participants 5, 6 and 7 with SARS-CoV-2.

Explants of oral or nasal tissue from participants 5, 6 and 7 were infected with SARS-CoV-2, strain IC19 as per the protocol described in Fig 1A. After 24 hrs of infection the tissue was washed and incubated for a further 24 hrs before transferring the tissue onto sponge rafts. At various time points supernatants were harvested over a 15-day period for assessment of viral replication of SARS-CoV-2 by qRT-PCR. Each experiment was set up in triplicate where possible depending on the size of the tissue received. The error bars represent the standard deviation between experiments. The dotted line represents the limit of detection (50 copies / reaction). NV = neat virus, Control = uninfected explants, High = high titre virus, Medium = 10-fold dilution of the high titre virus, Low = 10-fold dilution of the medium titre virus and 20 hrs = sample collected pre-wash of explants. For participant 5, a Mann Whitney t-test was used to determine significance between RNA transcripts detected at days 1–15, the resulting p-values are shown on top of each timepoint, ns = not significant. (TIF)</p

<i>Ex vivo</i> challenge of oral and nasal tissue from study participants 8, 9 and 10 with SARS-CoV-2.

Explants of oral or nasal tissue from participants 8, 9 and 10 were infected with SARS-CoV-2, strain IC19 as per the protocol described in Fig 1A. After 24 hrs of infection the tissue was washed and incubated for a further 24 hrs before transferring the tissue onto sponge rafts. At various time points supernatants were harvested over a 15-day period for assessment of viral replication of SARS-CoV-2 by qRT-PCR. Each experiment was set up in triplicate where possible depending on the size of the tissue received. The error bars represent the standard deviation between experiments. The dotted line represents the limit of detection (50 copies / reaction). NV = neat virus, Control = uninfected explants, High = high titre virus, Medium = 10-fold dilution of the high titre virus, Low = 10-fold dilution of the medium titre virus and 20 hrs = sample collected pre-wash of explants. (TIF)</p

S2 File -

With the onset of COVID-19, the development of ex vivo laboratory models became an urgent priority to study host-pathogen interactions in response to the pandemic. In this study, we aimed to establish an ex vivo mucosal tissue explant challenge model for studying SARS-CoV-2 infection and replication. Nasal or oral tissue samples were collected from eligible participants and explants generated from the tissue were infected with various SARS-CoV-2 strains, including IC19 (lineage B.1.13), Beta (lineage B.1.351) and Delta (lineage B.1.617.2). A qRT-PCR assay used to measure viral replication in the tissue explants over a 15-day period, demonstrated no replication for any viral strains tested. Based on this, the ex vivo challenge protocol was modified by reducing the viral infection time and duration of sampling. Despite these changes, viral infectivity of the nasal and oral mucosa was not improved. Since 67% of the enrolled participants were already vaccinated against SARS-CoV-2, it is possible that neutralizing antibodies in explant tissue may have prevented the establishment of infection. However, we were unable to optimize plaque assays aimed at titrating the virus in supernatants from both infected and uninfected tissue, due to limited volume of culture supernatant available at the various collection time points. Currently, the reasons for the inability of these mucosal tissue samples to support replication of SARS-CoV-2 ex vivo remains unclear and requires further investigation.</div

<i>Ex vivo</i> challenge of oral and nasal tissue from study participants 12 and 13 with Wuhan, Beta and Delta SARS-CoV-2 strains.

Explants from participants 12 and 13 were infected with different titres of either the IC19, Beta or Delta strain of SARS-CoV-2 as per the protocol described in Fig 1A. For each viral titre the experiment was set up in triplicate where possible. Supernatants were harvested at various time points for assessment by qRT-PCR for viral replication. Each experiment was set up in triplicate where possible depending on the size of the tissue received. The error bars represent the standard deviation between experiments. The dotted line represents the limit of detection (50 copies / reaction). NV = neat virus, Control = uninfected explants, High = high titre virus, Medium = 10-fold dilution of the high titre virus, and 20 hrs = sample collected pre-wash of explants. The solid bars represent IC19, the stripe bars represent the Beta strain and the dotted bars represent the Delta strain. The samples collected at days 7, 11 and 15 were not analyzed by qRT-PCR. (TIF)</p

<i>Ex vivo</i> challenge of oral and nasal tissue from study participants 14, 15 and 16 with SARS-CoV-2.

Two or four oral or nasal tissue explants from participants 14, 15 and 16 were infected with SARS-CoV-2, strain IC19 using the modified protocol as described in Fig 2A. After 2 hrs of infection the explants were washed and incubated for 48 hrs in liquid media. Culture supernatants were collected at 24 hrs and 48 hrs for viral replication assessment by qRT-PCR. Each experiment was set up in triplicate where possible depending on the size of the tissue received. The error bars represent the standard deviation between experiments. The dotted line represents the limit of detection (50 copies / reaction). NV = neat virus, Control = uninfected explants. (TIF)</p

Primers and probe sequences (with modifications) for variant calling and cDNA synthesis of Wuhan, Beta and Delta lineages of SARS-CoV-2.

Primers and probe sequences (with modifications) for variant calling and cDNA synthesis of Wuhan, Beta and Delta lineages of SARS-CoV-2.</p