TaffiX® nasal powder spray forms an effective barrier against infectious new variants of SARS-CoV-2 (COVID-19)

2021 ◽  
Author(s):  
Michal Mandelboim ◽  
Ella Mendelson ◽  
Yaron Drori ◽  
Nofar Atari ◽  
Tair Lapidot ◽  
...  
Keyword(s):  
South African ◽  
Rna Virus ◽  
Rna Extraction ◽  
Real Life ◽  
Preclinical Study ◽  
Viral Rna ◽  
Pcr Analysis ◽  
Effective Barrier ◽  
Gel Layer

Abstract Introduction: While vaccination efforts against SARS-CoV-2 around the world are ongoing -, new high-infectious variants of the virus are being detected. The protection of the available vaccines against some of the new variants is weaker, and experts are concerned that newer as yet undescribed variants of this mutated RNA virus will eventually prove stable against the current vaccines. Additional preventive measures will therefore be needed to protect the population until effective vaccinations are widely available.TaffiX® is a personal, anti-viral nasal powder spray comprised of low pH Hypromellose that upon insufflation into the nose creates a thin gel layer covering the nasal mucosa and forming a protective mechanical barrier that prevents viruses from engaging with nasal cells- the main portal of entry for viruses. Taffix is commercially available in many countries across Europe, Asia America and Africa. In a prior preclinical study, TaffiX® was found to be effective against SARS-CoV-2 Hong Kong/VM20001061/2020 in experimental in vitro conditions. A real-life clinical survey demonstrated that TaffiX® nasal spray significantly reduced the SARS-CoV-2 infection rate post mass-gathering event in a highly endemic community.Objective: The current study aimed to test the protective effect of Taffix against new pathogenic, highly infectious SARS-CoV-2 variants in vitro: the “British” B.1.1.7 (hCoV-19/Israel/CVL-46879-ngs/2020) and the “South African” B.1.351 (hCoV-19/Israel/CVL-2557-ngs/2020) variants.Study design: A TaffiX® gel was formed on a nylon filter, using an amount equivalent to a clinical dose of Taffix . Filters were then seeded with SARS-CoV-2 B.1.1.7 (“British”) and B.1.351 (“South African”) variants. After a 10 -minute incubation at room temperature, the bottom of each filter was washed, and the resulting flow-through was collected and seeded into 24 -well plates containing Vero-E6 cells. After 5 days of incubation, a 200 µl sample from each well was taken for viral RNA extraction followed by SARS-CoV 2 RT-PCR analysis.Results: The TaffiX® gel completely blocked SARS-CoV-2 highly infectious variants B.1.1.7 and B.1.351 in vitro, reducing the titer of recoverable infectious virus as well as viral RNA by 100%.Conclusions: Under in vitro conditions, TaffiX® formed an effective protective barrier against SARS-COV-2 variants (British variant and South African Variant). These results are consistent with prior findings demonstrating the in vitro high efficacy of Taffix gel in preventing viruses from reaching cells and infecting them. These results, added to clinical real-life studies performed with Taffix , support its use as an effective barrier against new variants of SARS-CoV-2 in conjunction with other protective measures.

Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Suppression of Viral RNA Recombination by a Host Exoribonuclease

2006 ◽  
Vol 80 (6) ◽  
pp. 2631-2640 ◽  
Author(s):  
Chi-Ping Cheng ◽  
Elena Serviene ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Rna Turnover ◽  
Rna Recombination ◽  
Viral Rnas ◽  
Yeast Strains ◽  
Genome Wide ◽  
Host Genes

ABSTRACT RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5′-3′ exoribonuclease, whose absence led to an ∼10- to 50-fold enhancement of RNA recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5′-truncated viral RNAs in xrn1Δ mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1Δ mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5′-truncated viral RNAs were expressed in the parental and xrn1Δ mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5′-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA recombination by rapidly removing the 5′-truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5′-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus recombination and evolution.

scholarly journals Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase

2005 ◽  
Vol 79 (16) ◽  
pp. 10608-10618 ◽  
Author(s):  
Zivile Panaviene ◽  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Replication Proteins ◽  
Rna Sequences ◽  
Alternative Structures ◽  
Recognition Element ◽  
Rna Elements

ABSTRACT Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3′-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

scholarly journals Optimized method for RNA extraction from leaves of forest tree species

2021 ◽  
Vol 875 (1) ◽  
pp. 012008
Author(s):  
T Grodetskaya ◽  
O Fedorova ◽  
P Evlakov
Keyword(s):  
Ammonium Acetate ◽  
High Efficiency ◽  
Rna Extraction ◽  
Forest Tree ◽  
Pcr Analysis ◽  
Ammonium Bromide ◽  
Qualitative And Quantitative Pcr ◽  
High Integrity ◽  
Forest Plants

Abstract Extraction of ribonucleic acid (RNA) from woody plants is a difficult task due to the peculiarities of plant material rich in polysaccharides and starch. The available techniques are often ineffective, since they result in the absence/reduced quality/reduced amount of RNA in the final preparation. The method we have optimized is based on the use of cethyltrimethyl ammonium bromide (CTAB), purification by phenol-chloroform extraction, use of lithium chloride and ammonium acetate. The method showed high efficiency for the extraction of RNA from the leaves of birch and poplar samples, in vitro and mature plants, in comparison with previously used methods (extraction using NucleoSpin® RNA Plant (Macherey-Nagel, Germany) columns, Su (2009) method, standard guanidine thiocyanate method). Electropherograms of RNA preparations showed its high integrity and concentration (up to 85 ng/μl), significantly higher purity of the preparation (up to 2.7 times). Purification of the preparation in the process of extraction can significantly reduce the yield of desoxyribonucleic acid (DNA). The optimized method is highly reproducible and can be used for further research, complementary DNA (cDNA) synthesis, qualitative and quantitative PCR analysis. The method allows obtaining high-quality RNA from other objects of agricultural and forest plants.

scholarly journals Purification of the Cucumber Necrosis Virus Replicase from Yeast Cells: Role of Coexpressed Viral RNA in Stimulation of Replicase Activity

2004 ◽  
Vol 78 (15) ◽  
pp. 8254-8263 ◽  
Author(s):  
Zivile Panaviene ◽  
Tadas Panavas ◽  
Saulius Serva ◽  
Peter D. Nagy
Keyword(s):  
De Novo ◽  
Rna Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Highly Active ◽  
Replicase Complex ◽  
Internal Initiation ◽  
The Stability

ABSTRACT Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for Cucumber necrosis tombusvirus (CNV), which is a plus-stranded RNA virus, from Saccharomyces cerevisiae. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), including the ability (i) to initiate cRNA synthesis de novo on both plus- and minus-stranded templates, (ii) to generate replicase products that are shorter than full length by internal initiation, and (iii) to perform primer extension from the 3′ end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.

scholarly journals Staufen1 Protein Participates Positively in the Viral RNA Replication of Enterovirus 71

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 142 ◽  
Author(s):  
Young-Mao Chen ◽  
Bo-Ting Ou ◽  
Chao-Ying Chen ◽  
Han-Hsiang Chan ◽  
Chih-Jung Chen ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Virus ◽  
Rna Stability ◽  
Enterovirus 71 ◽  
Viral Rna ◽  
Viral Translation ◽  
Rna Translation ◽  
Rna Accumulation

The double-stranded RNA-binding protein Staufen1 (Stau1) has multiple functions during RNA virus infection. In this study, we investigated the role of Stau1 in viral translation by using a combination of enterovirus 71 (EV-A71) infection, RNA reporter transfection, and in vitro functional and biochemical assays. We demonstrated that Stau1 specifically binds to the 5′-untranslated region of EV-A71 viral RNA. The RNA-binding domain 2-3 of Stau1 is responsible for this binding ability. Subsequently, we created a Stau1 knockout cell line using the CRISPR/Cas9 approach to further characterize the functional role of Stau1’s interaction with viral RNA in the EV-A71-infected cells. Both the viral RNA accumulation and viral protein expression were downregulated in the Stau1 knockout cells compared with the wild-type naïve cells. Moreover, dysregulation of viral RNA translation was observed in the Stau1 knockout cells using ribosome fractionation assay, and a reduced RNA stability of 5′-UTR of the EV-A71 was also identified using an RNA stability assay, which indicated that Stau1 has a role in facilitating viral translation during EV-A71 infection. In conclusion, we determined the functional relevance of Stau1 in the EV-A71 infection cycle and herein describe the mechanism of Stau1 participation in viral RNA translation through its interaction with viral RNA. Our results suggest that Stau1 is an important host factor involved in viral translation and influential early in the EV-A71 replication cycle.

scholarly journals The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

2014 ◽  
Vol 89 (5) ◽  
pp. 2750-2763 ◽  
Author(s):  
K. Reddisiva Prasanth ◽  
Daniel Barajas ◽  
Peter D. Nagy
Keyword(s):  
Viral Replication ◽  
Genetic Recombination ◽  
Rna Virus ◽  
Viral Rna ◽  
Rna Recombination ◽  
Viral Recombination ◽  
Antiviral Responses ◽  
New Hosts

ABSTRACTRNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination inSaccharomyces cerevisiaeand plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast orin vitrobased on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92polreplication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed inrpn11mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs.IMPORTANCERNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, andin vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination.

scholarly journals Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2162-2169 ◽  
Author(s):  
Saulius Serva ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Rna Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Mass Spectrometry Analysis ◽  
Host Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Viral Replicase

ABSTRACT Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown ∼35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.

scholarly journals Revealing the density of encoded functions in a viral RNA

2015 ◽  
Vol 112 (7) ◽  
pp. 2227-2232 ◽  
Author(s):  
Nikesh Patel ◽  
Eric C. Dykeman ◽  
Robert H. A. Coutts ◽  
George P. Lomonossoff ◽  
David J. Rowlands ◽  
...  
Keyword(s):  
Coat Protein ◽  
Self Assembly ◽  
Rna Virus ◽  
Viral Rna ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Viral Yield ◽  
Recognition Motifs ◽  
Packaging Signals

We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid self-assembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens.

scholarly journals P–793 Validation of French in vitro fertilization (IVF) guideline during Covid–19 pandemic by the research of Sars-Cov–2 RNA in the follicular fluid (FF) after egg retrieval

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Fossard ◽  
E Farfour ◽  
A Benammar ◽  
M Filali ◽  
J Vandame ◽  
...  
Keyword(s):  
Medical Practice ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Vitro Fertilization ◽  
Systematic Testing ◽  
Symptom Questionnaire ◽  
Health Authorities ◽  
Epidemiological Evaluation

Abstract Study question Is it possible to find viral Sars-Cov–2 RNA in FF of women undergoing treatment during Covid–19 pandemic that may compromise gamete and embryo safety? Summary answer No viral RNA was detected in tested FF of women undergoing IVF in compliance with recommendations. This was reassuring and supported good medical practice. What is known already Risks due to SARS-CoV–2 during IVF remain difficult to assess despite the screening recommended by French health authorities based on a symptom questionnaire of the couple (systematic testing by RT-PCR for the virus before egg retrieval (ER) is not mandatory). In this context, this is a real challenge for IVF laboratory to guarantee procedure, patients, gametes and embryos safety. Most studies have reported the absence of virus in sperm. No data are available for FF and only one study looked for the presence of the virus in oocytes of Covid-affected patients (Barragan M et al, 2020). Study design, size, duration Between June 17 and September 24, 2020, FF of consenting women were prospectively collected and symptom questionnaire recorded. During this period, women undergoing IVF in our center did not benefit from systematic PCR testing for the virus within 72 hours prior to ER through our health authorities’ recommendations. All collected FF were retrospectively tested to research viral RNA by RT-PCR and patients were recalled to answer an epidemiological follow-up questionnaire. Participants/materials, setting, methods For all couples, symptom questionnaires were prospectively recorded and verified at each step of IVF procedure. For all consenting women, a sample of 1 ml of FF was collected the day of ER and stored at –80 °C. After thawing, a Sars-Cov2 multiplex RT-PCR using CFX96 (Biorad*) was performed, after RNA extraction using Nimbus (Seegene*). A comprehensive epidemiological evaluation was made afterwards by phone interview and data were recorded and analyzed. Main results and the role of chance A total of 183 women was included out of the 214 treated during this period (85.5%). Retrospective epidemiological evaluation showed that 8 patients contracted Covid more than 2 months before the ER, 6 more than 2 months after and only one patient 1 month after ER (diagnosis based on pathognomonic signs as agueusia and anosmia or/and positive PCR ). We observed a prevalence of symptomatic Covid forms in our IVF population of 8.2% during a 6-month period surrounding their IVF cycle. Moreover, until the introduction of systematic testing by RT-PCR for the virus before ER since the end of September 2020, 3 patients have been cancelled out of the 403 planned for positive PCR despite a negative questionnaire, which represents a prevalence of asymptomatic forms on the day of the ER at 0.7%. All the 183 FF tested did not reveal any viral RNA detection, which was reassuring concerning our medical practice and patient compliance and transparency. The absence of detected viral RNA may be due to several reasons: 1) women were not infected the day of ER 2) women had an asymptomatic form of the disease with low viral load 3) FF is not a virus reservoir. Limitations, reasons for caution Not all patients were included (85.5%). Post-diagnosis stays uncertain because PCR tests at the beginning of the epidemic were not mandatory and hardly available. Wider implications of the findings: The absence of viral RNA in FF of women only screened through a symptom questionnaire is reassuring concerning the safety of IVF during Covid pandemic. Trial registration number Not applicable

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