Other replicas consist of proteins that facilitate viral replication and block the intrinsic host immune system by controlling the cellular machinery. In contrast to Nsp12, Nsp8 has primase capacity that makes it needless of any primers to initiate the viral replication process. In addition to these polymerases, the SARS-CoV-2 has a unique multimeric RNA polymerase composed of Nsp7 and Nsp8, which is responsible for both the initiation and elongation of the newly synthesized fragment of the viral genome [ 16 , 23 ].
From entering the host cells to virus particle formation, various viral processes are dependent on these proteins Fig. The spike or S glycoprotein is a transmembrane protein that resides on the outer surface of the virus, with nearly kDa molecular weight that determines the host range and pathogenicity of the virus by its contribution to viral entry through mediating the attachment of the virus particle to the plasma membrane of the host cell.
However, in some circumstances, they might use CDL as an alternative receptor. These viruses spread between different cell types with the help of this mechanism that suppresses virus-neutralizing antibody responses [ 16 , 21 , 25 ]. Membrane or M protein determines the shape of the virus envelope. It can function as a small transmembrane protein through binding to all other structural proteins. The M protein also contributes to the packaging of the viral RNA genome into a helical ribonucleocapsid during virion formation [ 16 ].
This protein takes part in various processes related to the viral genome, including the replication cycle of viruses, viral genome signaling, and the host cells' response to viral infections [ 22 , 25 ].
The structural evidence shows that the N-amino terminal of the envelope or E protein is a short hydrophilic sequence consisting of 7—12 amino acids. It is followed by a large hydrophobic middle segment with 25 amino acids known as the transmembrane domain and possesses a long hydrophilic C-terminus which constitutes the most of the protein.
E protein is involved in the production and maturation processes of the virus through interacting with the host cell membrane protein [ 22 ]. Spike protein uses a membrane-bound aminopeptidase called ACE2 as the primary cell receptor that binds to dipeptidyl peptidase-4 on the virus [ 27 ]. ACE2 is widely expressed in the various organs, including the heart, lungs, gastrointestinal tract, and kidneys [ 27 ].
Signal transmission through ACE2 is disrupted when severe myocardial damage and dysfunction in the lungs and other organs such as the kidneys and heart are induced when the virus attaches to the ACE2 receptor [ 28 ].
Besides, the determination of target cell specificity is influenced by the S glycoprotein interaction with cellular receptors. The location of the receptor binding domain RBD in S1 domain of S protein is determined by the structure of each virus, which can be variable.
SARS-CoV-2 uses some peptidases as their cellular receptors, although their entry occurs even in the absence of secondary enzymes [ 25 , 27 ]. After fastening to the receptor, the virus enters the host cell cytosol by transmembrane protease serine 2 TMPRSS2 or other proteases such as furin, and fuses the viral and cell membranes.
The furin detection location is crucial for detection by pyrolysis and, thus, in viral infection of the virus. This glycoprotein is broken down by furin proteinase into a subunit S1 and S2 like a host cell. The S2 domain is a viral fusion protein and is involved in a mixture of virion glands. Immediately after the virus entrance to the cell, the S glycoprotein is cleaved by the CTSL cathepsin to kill the S2 fusion peptide, and then forms endocytosis and low-pH endosomes, hence activating the fusion of membranes within the endosomes.
At the junction between the virus membrane and the cell membrane, a bundle is formed that is likely to release the virus genome into the cytoplasm. The following step in the infection cycle is the translation of replica genes from the RNA virus. The two polymer proteins pp1a and pp1 ab are encoded by two large proteins, ORF rep1a and rep1b. Usually, the ribosome opens the pseudoknot structure and continues the translation process until it encounters a pause or reach the end of 11 repetitions in the codon.
But sometimes, the ribosome is stopped by the pseudoknot, which prevents stretching and staying in the slippery sequence, and stops the nucleotide reading frame with a backward shift that results in the translation of pp1ab.
After the translation process, viral RNA is synthesized, and viral replication complexes are collected. The set of viral replications by the transcription process results in cytopathic effects such as abnormalities in the vascular complexes. In the downstream region of the ridiculous proteins, subgenomic RNAs act as mRNAs for the structural genes of the virus [ 29 ]. The next step is the binding and translating the of structural glycoproteins of virus, including S, M and E to ER.
Here, the viral genomes that encode structural proteins are encapsulated by the N-germ protein in the ERGIC membrane and become mature viruses. Protein-protein interaction is mainly mediated by M glycoprotein, which is responsible for collecting CoVs. M protein, expressed with E protein, is involved in the formation of virus-like particles VLPs as well as in the formation of coatings for CoVs.
Because M protein is abundant compared to E protein, M protein interaction can be a major source of motivation to mask puberty. Glycoprotein changes the host secretion pathway and releases the accumulation of the virus in the host.
The M protein attaches to the nucleocapsid located at the C-terminus of endo domain M and signals viral assembly completion. After assembly, the viruses are transmitted by vesicles to the host cell membrane and are released by exocytosis. By being transported to the cell surface, the S protein combines cell fusion between infected and healthy cells, resulting in a large multinucleated cell that spreads the virus in the host and provides the conditions for the detection of specific antibodies against the virus Fig.
The mechanism of coronavirus infection and spread. Coronaviruses bind to ACE-2 receptors on the surface of the target cell via their spike proteins. Besides, variations may appear in the course of each genome replication cycle [ 30 ].
It can be used for evolutionary investigations, being useful in identifying mutations in the coronavirus genome, where many mutations may occur by cause of an RdRp in the process of genome replication [ 31 , 32 ]. Table 1 and Fig. This paper recognized that these four mentioned-mutations are common in the SARS-CoV-2 European isolates genomes, where the severity of the infection is mostly more intense than in the other geographical regions.
The nsp3 collaborates with two other proteins called nonstructural protein-4 and 6, to bring about DMVs, which are complexes of the membrane that assist in replicating and assembling RNA.
Mutations in RNA polymerase or RNA primase might increase fidelity and present obstruction to the usage of nucleotide analogs that are mutagenic. ORF1ab gene, which has the most length in the ORFs in the virus genome and is split into viral non-structural proteins nsp1 to nsp16 , was reported as a gene with a high number of missense mutations [ 23 , 37 ]. After analyzing around 48, SARS-CoV-2 genome sequences and comparing them with the reference sequence, researchers demonstrated that there reside at least three clades according to geological and genomic relevance.
The clade common in Europe obtains the p. The other significant mutation was in the N protein p. RGKR [ 34 ]. Based on a study that analyzed genome sequences from COVID patients, the scientists appraised the disposition of the virus mutations within four geographic regions by standardizing the number of genomes containing a likely mutation in each region. The appearance of mutations in nucleotide locations , , , , and has been confirmed.
Besides, mutations were found in positions , , , , , , , , existing in sequences of ORF-1ab , , , , , and , S and N One mutation location causes the substitution of 2 amino acids, particularly Arg to Lys and Gly to Arg [ 38 ]. According to a paper that has analyzed the SARS-CoV-2 genomes, 10 out of the 48 sequences merely had lost their base pairs at the start and the end. However, 80 alternatives contained mutations including, 43 missenses, 21 synonymous, three deletions, and 13 noncoding deletion types.
Out of the 43 missense alternatives, 30 ones were observed in ORF-1ab. All three deletion mutations found in this study, are in-frame ones and were observed in nsp1, which is in ORF1ab. L84S , is not conserved [ 36 ]. Khailany et al. Wang et al. They compared the isolates with the reference sequence and depicted that the total homology was Also, the homology of the ORF-1a in the genome was The results show that by comparing the amino acids of the sequences, the overall homology was Mutations in amino acid sequences appearing in more than three strains were p.
Besides, six deletion mutations were observed in 5 of the sequences. These mutations led to 4 various shortened variations in the sequence of amino acids. Yu et al. Multiple sequence alignment of 86 patient-isolated strains of the SARS-CoV-2 disclosed 93 mutations including 42 missense mutations in viral proteins except for the E protein, 29 missense mutations in the ORF1ab protein, and 8 of them in the S protein [ 40 ].
In another paper, ten viral genomes derived out of the NCBI database were analyzed through sequence alignment. They did not see any divergence in the amino acid arrangement of N and M proteins. Two alternatives in terms of amino acid level, in spike protein, were detected. Holland et al. Unlike other methods, it does not involve computer simulations, complex mathematical theories, or calculus.
The purpose of this protocol is to provide a detailed outline of the method along with the parameter optimizations needed for a successful assay. The basic idea of the fluctuation analysis is to start growing many replicate cultures of cells that initially have no mutation of interest, to let them go through a certain number of cell divisions to saturation—so as to limit the number of generations—in a permissive medium, and to measure how many of the cultures have not acquired the mutations.
There are two successive selections: the first one selects for absence of the mutation while the second one selects for presence of the mutation. The cells are then grown in permissive condition for a defined window of time during which mutations may accumulate.
By subsequently selecting only those cells that have acquired the mutations, it is easy to exclude cells that have failed to obtain the mutation—those cells cannot form colonies. When growing multiple replicate cultures, the permissive medium must provide conditions where the mutation is neither selected for nor selected against i. The number of cell divisions that occurred in each replicate culture can be estimated by measuring the final cell population of the culture.
Knowing the total number of cell divisions and the number of cultures that failed to give colonies in the second selection i. Therefore, it is critical to identify optimal culture conditions and thus the optimal culture size that will give rise to the appropriate number of cellular generations at saturation so as to yield a fraction of zero-mutation events that is in the desired range.
There are numerous ways to control the number of cell divisions that occur in a culture by the time it reaches saturation:. Many different combinations of these factors can be used to get the correct size of zero-class events.
Because the mutation rate can differ between mutation types, strains, and experimental conditions, you may have to repeat this optimization step several times.
Four different conditions that were tested for use in an actual experiment are shown below; only the dextrose concentration was varied:. Grow an overnight culture of the strain to be tested in medium that selects against growth of cells in which the mutation has already occurred.
Obtain cell densities in 10 out of 48 wells for each condition and make sure that cell counts vary as expected with the dextrose concentrations. Sonicate before counting. Mix the contents of the wells before spotting because cells on the bottom of each well will not readily come out. Place the entire volume of each culture onto a dried selective plate. Incubate the selective plates for a pre-determined amount of time at the right temperature for your strain until visible and countable colonies appear.
Calculate the fraction of zero-class events. Grow an overnight culture of the strain to be tested in medium that selects against growth of cells with the mutation. To do this test, directly dispense from the large volume mixture an appropriate number of cells for counting colonies on permissive plates. The count of colony-forming units will be later used as the initial cell number for each well, which is useful for calculating how many generations of growth have occurred. This test will also account for any cell misbehavior, measurement error, dilution error, etc.
We examined three porous objects tissue, wood, and porous glass that were sufficiently hydrophilic that the droplet could penetrate into the solid.
Each of these porous solids had less transfer than each of the three non-porous solids glass, stainless steel, or Teflon. This is evident for wet and dried solids considered separately Fig. The most direct comparison is between porous and non-porous glass, where the material is the same yet the transfer is completely different.
The explanation is fairly simple. For wet surfaces, the droplet lies mainly within the porosity at the time of contact with the skin, and so less of the droplet is transferred to skin. For dried droplets, we expect that much of the virus is dried into the interior of the porosity and is not available on the surface for dry, solid-to-solid transfer.
We did some further tests on the effect of porosity using real time quantitative polymerize chain reaction RT-qPCR measurements. We expect that the effect of porosity would be contingent on the interior pores of the solid being wettable.
Thus, we would not expect the droplet to penetrate porous Teflon, so we predict transfer would still be high from this material. That work showed that transfer was greater from plastic than from cardboard.
The authors state that the plastic was non-porous and that the cardboard was porous, and make a similar conclusion that the lack of transfer for cardboard is due to the drop being absorbed into the porous surface. The paper material provides an interesting example of a porous material. Transfer from paper was initially after 10 s very high whereas transfer after the droplet evaporated 30 min was very low.
This is explained though an understanding of how the wettability of the material affects access to the porosity. If the chemistry of pores is sufficiently hydrophobic, then the water cannot spontaneously enter the pores and the porosity is not accessed.
Although paper is porous, there is an induction time for wetting: for the particular paper that we used, the paper goes from non-wetting and impermeable to wetting and permeable after about 2. Without access to the porosity, the SARS-CoV-2 suspension behaved as if it were on a non-porous solid, the droplet was transferred to the finger and we recorded a high transfer ratio.
After 2. The transfer experiment after 30 min showed a very low level of transfer because those interior surfaces were not accessible to the finger during transfer. We also measured the TCID 50 of liquid used to extract virus from the dried solids after contact with the finger data not shown using the same procedure as for extraction from the skin see Supplementary Information S.
We were not surprised that the levels were very low for porous solids. This model performed well and is described in Supplementary Fig. People use a huge range of contact trajectories when touching surfaces, for example, different pressures, different times of contact, and different rubbing actions.
In this study we considered only brief and low-pressure contact with no rubbing or translation in contact. It is well-established that rubbing causes friction and increases wear and transfer between solids, and in particular that microbial transfer is increased significantly by rubbing For example, Pan et al. This still leaves a large dose of virus on the finger. Clearly one more step is required to transfer the virus to the respiratory system: transfer from the finger to the nose or mouth.
The overall transmission from solid to skin to respiratory system depends on the product of the individual transfer rates, and would be lower. The final dose transferred to the respiratory system should be compared to the infectious dose for a human, which unfortunately is not known. Studies of Syrian hamsters indicate that the infective does for these susceptible animals is only 5 infectious particles Clearly hand washing can reduce both the initial contamination of surfaces and intercept the virus before it is transferred from fingers to the respiratory system.
Infection via fingers depends also on the longevity of the virus on human skin: the virus must survive long enough on skin to be transferred. Harbourt et al. Hirose et al. These longevity studies suggest that once SARS-CoV-2 has been transferred to the skin, there is a long period where there is an opportunity to transfer viable virus to the eye, nose, or mouth. In summary, our research demonstrates substantial transfer of SARS-CoV-2 virus from a variety of solids to an artificial finger after a brief, light force and no rubbing.
The transfer is lower but still occurs after the droplet has dried. When the droplet penetrates a porous solid, such as wood or tissue, the transfer is low. The data that supports the findings of this study are available within the Supplementary Information. World Health Organization. Center for Disease Control. Cleaning and Disinfection for Households. Sia, S. Nature , — Meiksin, A. Van Doremalen, N. Article Google Scholar.
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