scholarly journals CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses?

2021 ◽  
Vol 15 (1) ◽  
pp. 32
Author(s):  
Diana Perez-SanJose ◽  
Miguel Angel de la Fuente ◽  
Julia Serna Pérez ◽  
Maria Simarro ◽  
José María Eiros Bouza ◽  
...  

Influenza viruses provide a great threat for the human population, causing highly contagious respiratory infections that can lead to serious clinical complications. There are a limited variety of influenza antivirals, and these antivirals are subjected to the constant emergence of resistances. Therefore, the development of new antiviral strategies to combat influenza viruses and other RNA viruses must be promoted. In this work, we design a proof-of-concept of a recently described CRISPR/Cas tool that has been proposed as a possible future RNA virus antiviral, named CRISPR/CasRx. For this, we verified the efficiency of the CasRx endonuclease in the degradation of the eGFP mRNA reporter gene and we established the best conditions for, and the efficient performance of, the CRISPR/CasRx system. The results were measured by fluorescence microscopy, flow cytometry, and qRT-PCR. The analyses demonstrated a reduction in fluorescence, regardless of the amount of eGFP reporter plasmid transfected. The analyses showed an 86–90% reduction in fluorescence by flow cytometry and a 51–80% reduction in mRNA expression by qRT-PCR. Our results demonstrate that the CasRx endonuclease is an efficient tool for eGFP mRNA knockdown. Therefore, subsequent experiments could be useful for the development of a new antiviral tool.

2018 ◽  
Vol 26 ◽  
pp. 204020661876448 ◽  
Author(s):  
Paul C Jordan ◽  
Sarah K Stevens ◽  
Jerome Deval

Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.


2021 ◽  
Author(s):  
Sorush Niknamian

Background: A virus is a small infectious agent that replicates only inside the living cells of an organism. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. In evolution, viruses are an important means of horizontal gene transfer, which increases genetic diversity in a way analogous to sexual reproduction. Influenza (Including (COVID-19), is an infectious disease caused by an influenza virus. Some viruses especially smallpox, throughout history, has killed between 300-500 million people in its 12,000year existence. As modern humans increased in numbers, new infectious diseases emerged, including SARS-CoV-2. We have two groups of virus, RNA and DNA viruses. The most brutal viruses are RNA ones like COVID-19 (Sars-CoV-2 [1] Introduction: Coronaviruses are a group of viruses that cause diseases in mammals and birds. In humans, coronaviruses cause respiratory tract infections that are typically mild, such as some cases of the common cold (among other possible causes, predominantly rhinoviruses), though rarer forms can be lethal, such as SARS, MERS, and COVID-19. Symptoms vary in other species: in chickens, they cause an upper respiratory tract disease, while in cows and pigs they cause diarrhea. Coronaviruses constitute the subfamily Orthocoronavirinae, The genome size, coronaviruses ranges from approximately 27 to 34 kilobases, the largest among known RNA viruses. Discussions and Results: We have researched from the first virus in the planet to the last mutated version which is SARS-COV-2. We have collected many informative data in tables and figures to reach the main cause of 2019Coronavirus and calculated the probability and estimated deaths in the current time. We have discussed about the possible treatment and prevention of the virus and did algebraic calculations on the epidemiology, the size and even the future of this pandemic. The only era which any virus had not been epidemic, were through world war 2, were the German scientists had found the way to fight any viral infections which is very important and can help scientists to reach the main treatment of the new 2019-Coronavirus. We have sorted the deadly and non-deadly coronaviruses and explained how this epidemic had begun through Evolutionary Medicine (EM). The result of the article is that 16% of the whole population in the world has been contaminated which is 1248000000 of 7.8 billion people world-wide. SARS-CoV-2 is an RNA Virus. its nucleic acid is 2 single-stranded RNA (ssRNA). The polarity of this virus is positive-sense ((+) ssRNA). Positivesense viral RNA is similar to mRNA and thus can be immediately translated by the host cell. Recombination in RNA viruses appears to be an adaptation for coping with genome damage. Recombination can occur infrequently between animal viruses of the same species but of divergent lineages. The resulting recombinant viruses may sometimes cause an outbreak of infection in humans. RNA viruses have very high mutation rates This is one reason why it is difficult to make effective vaccines to prevent diseases caused by RNA viruses. The resulting recombinant viruses causes an outbreak of infection in humans. Conclusion: In conclusion, the mutation of the SARSCoV and influenza viruses through Drift and Reassortment is the main cause of SARS-CoV-2 through natural selection, Lamarckian Evolution and coevolution which caused this RNA virus so powerful, unpredicted and different in the genome size and nations worldwide. The first Pandemic of Influenza was first detected in 1732 and this virus evolved through natural selection till 2019 which caused the worldwide pandemic of SARS-CoV-2. Based on many studies, inhalation of Ozone plus Sulfur Dioxide, increasing the amounts of L-Glutathione (Which is low in children and older adults and this is the main reason why older adults and children die from this disease.) plus Viral Phage Therapy (VPT) which we discussed fully in this article can be the possible prime treatment of SARS-CoV-2 infection. The seasonal temperature cannot be useful in controlling/reducing the pandemic of this virus since the natural selection, Lamarckian Evolution and high mutation of the virus helps its survival. No antiviral drugs will be useful against SARSCoV-2 because of high rate of mutation and primarily adaptation of the virus to the drugs and even the environmental Temperature.


2021 ◽  
Vol 18 (184) ◽  
Author(s):  
Harsh Chhajer ◽  
Vaseef A. Rizvi ◽  
Rahul Roy

Life cycle processes of positive-strand (+)RNA viruses are broadly conserved across families, yet they employ different strategies to grow in the cell. Using a generalized dynamical model for intracellular (+)RNA virus growth, we decipher these life cycle determinants and their dependencies for several viruses and parse the effects of viral mutations, drugs and host cell permissivity. We show that poliovirus employs rapid replication and virus assembly, whereas the Japanese encephalitis virus leverages its higher rate of translation and efficient cellular reorganization compared to the hepatitis C virus. Stochastic simulations demonstrate infection extinction if all seeding (inoculating) viral RNA degrade before establishing robust replication critical for infection. The probability of this productive cellular infection, ‘cellular infectivity’, is affected by virus–host processes and defined by early life cycle events and viral seeding. An increase in cytoplasmic RNA degradation and delay in vesicular compartment formation reduces infectivity, more so when combined. Synergy among these parameters in limiting (+)RNA virus infection as predicted by our model suggests new avenues for inhibiting infections by targeting the early life cycle bottlenecks.


2020 ◽  
Vol 63 (3) ◽  
pp. 7-18
Author(s):  
Marcela Solís-Rodríguez ◽  
Ángel G. Alpuche-Solís ◽  
Rocío G. Tirado-Mendoza

In 2001 in the Netherlands, Human metapneumovirus (hMPV) was identified as a “new” etiologic agent causing acute respiratory infections in children younger than 5 years old; however, it has also been isolated in the elderly and immunocompromised people. This virus is considered the second etiological agent in acute diseases of the respiratory tract. Currently, the estimated cost of IRAs in our country is of 9,000USD per inpatient. hMPV is a member of the genus Metapneumovirus, family Pneumoviridae, and it belongs to the order Mononegavirales that is part of the negative single-stranded ribonucleic acid (RNA) virus, consisting of eight genes ordered: 3’-N-P-M-FM2-SH-G-L-5 ‘, and which encodes for 9 proteins. Of these proteins, the F fusion glycoprotein is highly conserved in the genus Metapneumovirus, and is the major antigenic determinant, and because an approved vaccine doesn’t exist, it has been used as a candidate epitope for the design of a vaccine that confers host immunity or as a therapeutic target in the creation of antiviral peptides that inhibit the fusion of the virus to its target cell and to avoid infection in subjects at high risk of contagion since there is currently none accepted by COFEPRIS as a prophylactic treatment against hMPV. Key words: hMPV; respiratory infections; epitopes; protein F;vaccines.


Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 313
Author(s):  
Heli A. M. Mönttinen ◽  
Janne J. Ravantti ◽  
Minna M. Poranen

RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization.


Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 556
Author(s):  
Kacper Toczylowski ◽  
Magdalena Wietlicka-Piszcz ◽  
Magdalena Grabowska ◽  
Artur Sulik

The cold season is usually accompanied by an increased incidence of respiratory infections and increased air pollution from combustion sources. As we are facing growing numbers of COVID-19 cases caused by the novel SARS-CoV-2 coronavirus, an understanding of the impact of air pollutants and meteorological variables on the incidence of respiratory infections is crucial. The incidence of influenza-like illness (ILI) can be used as a close proxy for the circulation of influenza viruses. Recently, SARS-CoV-2 has also been detected in patients with ILI. Using distributed lag nonlinear models, we analyzed the association between ILI, meteorological variables and particulate matter concentration in Bialystok, Poland, from 2013–2019. We found an exponential relationship between cumulative PM2.5 pollution and the incidence of ILI, which remained significant after adjusting for air temperatures and a long-term trend. Pollution had the greatest effect during the same week, but the risk of ILI was increased for the four following weeks. The risk of ILI was also increased by low air temperatures, low absolute humidity, and high wind speed. Altogether, our results show that all measures implemented to decrease PM2.5 concentrations would be beneficial to reduce the transmission of SARS-CoV-2 and other respiratory infections.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
David R. McIlwain ◽  
Han Chen ◽  
Maria Apkarian ◽  
Melton Affrime ◽  
Bonnie Bock ◽  
...  

Abstract Background Influenza places a significant burden on global health and economics. Individual case management and public health efforts to mitigate the spread of influenza are both strongly impacted by our ability to accurately and efficiently detect influenza viruses in clinical samples. Therefore, it is important to understand the performance characteristics of available assays to detect influenza in a variety of settings. We provide the first report of relative performance between two products marketed to streamline detection of influenza virus in the context of a highly controlled volunteer influenza challenge study. Methods Nasopharyngeal swab samples were collected during a controlled A/California/2009/H1N1 influenza challenge study and analyzed for detection of virus shedding using a validated qRT-PCR (qPCR) assay, a sample-to-answer qRT-PCR device (BioMerieux BioFire FilmArray RP), and an immunoassay based rapid test kit (Quidel QuickVue Influenza A + B Test). Results Relative to qPCR, the sensitivity and specificity of the BioFire assay was 72.1% [63.7–79.5%, 95% confidence interval (CI)] and 93.5% (89.3–96.4%, 95% CI) respectively. For the QuickVue rapid test the sensitivity was 8.5% (4.8–13.7%, 95% CI) and specificity was 99.2% (95.6–100%, 95% CI). Conclusion Relative to qPCR, the BioFire assay had superior performance compared to rapid test in the context of a controlled influenza challenge study.


2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
Author(s):  
Himanshu Kumar ◽  
Taro Kawai ◽  
Hiroki Kato ◽  
Shintaro Sato ◽  
Ken Takahashi ◽  
...  

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.


2021 ◽  
Vol 22 (5) ◽  
pp. 2723
Author(s):  
Linhua Tian ◽  
Elzafir B. Elsheikh ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
Adolfas K. Gaigalas ◽  
...  

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


2008 ◽  
Vol 389 (10) ◽  
Author(s):  
Stephan Pleschka

AbstractThe Raf/MEK/ERK signal transduction cascade belongs to the mitogen-activated protein kinase (MAPK) cascades. Raf/MEK/ERK signaling leads to stimulus-specific changes in gene expression, alterations in cell metabolism or induction of programmed cell death (apoptosis), and thus controls cell differentiation and proliferation. It is induced by extracellular agents, including pathogens such as RNA viruses. Many DNA viruses are known to induce cellular signaling via this pathway. As these pathogens partly use the DNA synthesis machinery for their replication, they aim to drive cells into a proliferative state. In contrast, the consequences of RNA virus-induced Raf/MEK/ERK signaling were less clear for a long time, but since the turn of the century the number of publications on this topic has rapidly increased. Research on this virus/host-interaction will broaden our understanding of its relevance in viral replication. This important control center of cellular responses is differently employed to support the replication of several important human pathogenic RNA viruses including influenza, Ebola, hepatitis C and SARS corona viruses.


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