Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines

Translocation of Fasciola hepatica via international livestock movements: development of ante-mortem molecular diagnostic tools for the identification of Fasciola spp. in livestock

Fasciolosis caused by infection with Fasciola hepatica and Fasciola gigantica is a zoonotic disease of worldwide importance, with an estimated 91 million people considered at risk of infection and livestock losses expected to exceed US $3 billion/year. Despite the significant human and animal health impacts, no test is capable of ante-mortem Fasciola spp. differentiation in areas of parasite sympatry. The aim of this thesis was to design, validate and deploy a suite of highly sensitive molecular diagnostic tools for Fasciola spp. differentiation from faecal samples to enable ante-mortem screening of livestock in Northern Laos. In combination with a traditional sedimentation method and with the use of a high-speed benchtop homogeniser, the detection and quantification of Fasciola spp. infection in 100% of cattle with low faecal egg loads (<25 EPG) was possible. The point of first detection of F. hepatica infection in experimentally-infected sheep was then examined and compared to the sedimentation and a commercially-available coproantigen ELISA. Faecal samples were first considered positive at 6, 7 and 8 weeks post infection (WPI) by coproELISA, real-time PCR and sedimentation, respectively. A simplified method using un-concentrated faecal samples was developed to increase sample throughput. The limit of detection using this method was 10 and 20 EPG for sheep and cattle, respectively. Finally, several single nucleotide polymorphism assays were developed to differentiate Fasciola spp. in faecal samples alongside a Next Generation Sequencing method to determine the contribution of nucleotides from each species. These assays were applied to 153 faecal samples collected from local cattle across 27 villages in Northern Laos to detect F. hepatica translocation in an area of SE Asia with frequent international livestock trade. Of the 91 positive samples, 11 were identified as containing F. hepatica DNA, indicating establishment of the F. hepatica lifecycle in Northern Laos

Herminthology: promoting gender equity in science and parasitology.

Gender inequity in Science, Technology, Engineering, and Medicine (STEM) fields, including parasitology, continues to limit the participation of women in scientific leadership and development. Here we highlight the aims and activities of Herminthology, an initiative promoting the work of women in parasitology, alongside the current status quo of men and women scientists in the discipline

Correlation between faecal egg counts and real-time PCR egg estimates.

<p>(A) Real-time PCR egg estimates (qEPG) for three methods of sample preparation for DNA isolation from cattle samples collected during Austral spring (Herd 1) are compared to morphological FEC (mEPG); isolation of DNA from 150 mg raw faeces (Method 1), sedimentation followed by isolation of DNA from 150 μl of the resultant sediment (Method 2), and sedimentation followed by isolation of DNA from the entire sediment pellet (Method 3). (B) As for A using only Method 3 on samples collected during Austral summer (Herd 1). (C) As for B using samples collected from an endemic population (Herd 2). (D) Correlation between duplicate sedimentations (mEPG) for all samples (Herds 1 and 2) across all sampling periods; inset–correlation between duplicate FECs for samples with ≤ 20 EPG.</p

Scrambled eggs: A highly sensitive molecular diagnostic workflow for <i>Fasciola</i> species specific detection from faecal samples

<div><p>Background</p><p>Fasciolosis, due to <i>Fasciola hepatica</i> and <i>Fasciola gigantica</i>, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of <i>F</i>. <i>hepatica</i> prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of <i>Fasciola</i> spp. in faecal samples.</p><p>Methodology/Principal findings</p><p>A new molecular diagnostic workflow for the highly-sensitive detection and quantification of <i>Fasciola</i> spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R² = 0.74–0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65–88%), compared to the sensitivity (91–100%) of the new molecular diagnostic workflow.</p><p>Conclusions/Significance</p><p>Species-specific assays for sensitive detection of <i>Fasciola</i> spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.</p></div

Correlation between faecal egg counts and real-time PCR egg estimates.

<p>(A) Real-time PCR egg estimates (qEPG) for three methods of sample preparation for DNA isolation from cattle samples collected during Austral spring (Herd 1) are compared to morphological FEC (mEPG); isolation of DNA from 150 mg raw faeces (Method 1), sedimentation followed by isolation of DNA from 150 μl of the resultant sediment (Method 2), and sedimentation followed by isolation of DNA from the entire sediment pellet (Method 3). (B) As for A using only Method 3 on samples collected during Austral summer (Herd 1). (C) As for B using samples collected from an endemic population (Herd 2). (D) Correlation between duplicate sedimentations (mEPG) for all samples (Herds 1 and 2) across all sampling periods; inset–correlation between duplicate FECs for samples with ≤ 20 EPG.</p

C_T values of <i>F</i>. <i>hepatica</i> eggs in sheep faecal samples when subjected to three different disruption treatments.

<p>C_T values of <i>F</i>. <i>hepatica</i> eggs in sheep faecal samples when subjected to three different disruption treatments.</p

C_T values of samples subjected to different storage conditions prior to sedimentation and DNA isolation.

<p>C_T values of samples subjected to different storage conditions prior to sedimentation and DNA isolation.</p