Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex.

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.

COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/µL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.

Actionable wastewater surveillance: application to a university residence hall during the transition between Delta and Omicron resurgences of COVID-19.

Wastewater surveillance has gained traction during the COVID-19 pandemic as an effective and non-biased means to track community infection. While most surveillance relies on samples collected at municipal wastewater treatment plants, surveillance is more actionable when samples are collected "upstream" where mitigation of transmission is tractable. This report describes the results of wastewater surveillance for SARS-CoV-2 at residence halls on a university campus aimed at preventing outbreak escalation by mitigating community spread. Another goal was to estimate fecal shedding rates of SARS-CoV-2 in a non-clinical setting. Passive sampling devices were deployed in sewer laterals originating from residence halls at a frequency of twice weekly during fall 2021 as the Delta variant of concern continued to circulate across North America. A positive detection as part of routine sampling in late November 2021 triggered daily monitoring and further isolated the signal to a single wing of one residence hall. Detection of SARS-CoV-2 within the wastewater over a period of 3 consecutive days led to a coordinated rapid antigen testing campaign targeting the residence hall occupants and the identification and isolation of infected individuals. With knowledge of the number of individuals testing positive for COVID-19, fecal shedding rates were estimated to range from 3.70 log10 gc ‧ g feces-1 to 5.94 log10 gc ‧ g feces-1. These results reinforce the efficacy of wastewater surveillance as an early indicator of infection in congregate living settings. Detections can trigger public health measures ranging from enhanced communications to targeted coordinated testing and quarantine.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses.

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

Reverse Genetics System for Rabbit vesivirus.

Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5'-cap structure was added to the 5' end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

Electrochemical quantification of Ag2S quantum dots: evaluation of different surface coating ligands for bacteria determination.

In this work, novel silver sulphide quantum dots (Ag2S QD) are electrochemically quantified for the first time. The method is based on the electrochemical reduction of Ag+ to Ag0 at -0.3 V on screen-printed carbon electrodes (SPCEs), followed by anodic stripping voltammetric oxidation that gives a peak of currents at +0.06 V which represents the analytical signal. The optimized methodology allows the quantification of water-stabilized Ag2S QD in the range of approximately 2 × 109-2 × 1012 QD·mL-1 with a good reproducibility (RSD: 5%). Moreover, as proof-of-concept of relevant biosensing application, Ag2S QD are evaluated as tags for Escherichia coli (E. coli) bacteria determination. Bacteria tagged with QD are separated by centrifugation from the sample solution and placed on the SPCE surface for quantitative analysis. The effect of two different Ag2S QD surface coating/stabilizing agents on both the voltammetric response and the bacteria sensing is also evaluated. 3-mercaptopropionic acid (3-MPA) is studied as model of short length coating ligand with no affinity for the bacteria, while boronic acid (BA) is evaluated as longer length ligand with chemical affinity for the polysaccharides present in the peptidoglycan layer on the bacteria cells surface. The biosensing system allows to detect bacteria in the range 10-1-103 bacteria·mL-1 with a limit of detection as low as 1 bacteria·mL-1. This methodology is a promising proof-of-concept alternative to traditional laboratory-based tests, with good sensitivity and short time and low cost of analysis. Graphical abstractNovel silver sulphide quantum dots (Ag2S QD) are electrochemically quantified for the first time. Moreover, Ag2S QD are evaluated as tags for Escherichia coli bacteria determination. The effect of two different QD surface coating ligands is also evaluated.

Myxoma virus jumps species to the Iberian hare.

The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host-pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis-like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5-month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next-generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population.

Molecular characterization and expression analyses of the Solea senegalensis interferon-stimulated gene 15 (isg15) following NNV infections.

Interferons are essential in fish resistance to viral infections. They induce interferon-stimulated genes, such as isg15. In this study, the Senegalese sole isg15 gene (ssisg15) has been characterized. As other isg15, ssisg15 contains a 402-bp intron sited in the 5'-UTR, and the full length cDNA is 1492-bp, including a 480-bp ORF. The expression analyses revealed basal levels of isg15 transcripts, and a clear induction after poly I:C injection, that reached maximum values in brain, head kidney and gills. The ssisg15 induction patterns were similar in RGNNV- and SJNNV-inoculated fish, whereas the reassortant (RG/SJ) isolate, which has higher replication fitness, triggered delayed but higher transcript levels. Furthermore, RG/SJ infection after poly I:C treatment reduced the induction of ssisg15 transcripts, suggesting an antagonistic mechanism against interferon type I system, that might allow an efficient viral replication at the initial steps of the infective process.

Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.