Visible morphological changes in cells caused by viral infection are called cytopathic effects CPE ; the responsible virus is said to be cytopathogenic.
The degree and types of visible damage to cells caused by viral infection vary greatly. Via antibodies Firstly, the antibodies neutralise the virus , meaning that it is no longer capable of infecting the host cell. Secondly, many antibodies can work together, causing virus particles to stick together in a process called agglutination. Viruses make us sick by killing cells or disrupting cell function. Our bodies often respond with fever heat inactivates many viruses , the secretion of a chemical called interferon which blocks viruses from reproducing , or by marshaling the immune system's antibodies and other cells to target the invader.
These are: 1 attachment; 2 penetration; 3 uncoating; 4 replication; 5 assembly; 6 release. As shown in , the virus must first attach itself to the host cell. This is usually accomplished through special glycoprotiens on the exterior of the capsid, envelope or tail.
Antibiotics can treat bacterial infections , such as: Most sinus infections. Strep throat. Urinary tract infections. Most ear infections otitis media Nasty bacterial skin infections impetigo. To reduce the infection of infectious diseases, following precautions can be taken in schools: Drinking clean and hygienic water. Virus concentration — It is important to perform a viral titer experiment prior to the CPE assay to determine the proper range of the virus concentrations in order to obtain the appropriate TCID50 results.
Throughput — Utilizing the plate-based image cytometry method, the assay throughput can be significantly increased from the standard 6 — well plates to 96 and well plates.
Imaging — Image cytometry can rapidly scan whole wells of the entire plate to digitally capture bright field and fluorescent images for analysis. Unlike the conventional microscopy method, which requires manual observation of the CPE, and lacks digital records. Analysis — Image analysis algorithms can automate the identification of CPE via the destruction of cell monolayer, reduction in host cell count, and morphological changes at an individual cell level.
In contrast, traditional CPE assays require trained technicians to visually inspect and identify, which is tedious, time-consuming, and generate a high level of uncertainties. Directly analyze cytopathic effect using the Celigo Image Cytometer The Celigo Image Cytometer is a sophisticated plate imager that can rapidly image the entire microplate in bright field and fluorescence.
Measure the host cell monolayer using confluence application The pseudo-green color shows where cells are present and that area is quantified and compared between different viral treatments. Healthy cells left and infected cells right 2. Count the total number of cells in each well The green outlines identify the counted cells in the well. If correct and general, it may have important implications for understanding of innate immunity mechanisms as well as pathogenesis and treatment of viral diseases.
Some major features of the structure and reproduction of picornaviruses are briefly summarized in Box 1. In many picornavirus—cell combinations, infection terminates in death of the host cell.
Two major types of picornavirus-induced fatal CPE are usually discerned, necrosis, and apoptosis Box 2. It is not obvious, however, to what extent cellular injuries reflect direct needs of viral reproduction, on the one hand, or interaction between host defensive and viral antidefensive activities, on the other.
Indeed, certain picornaviruses, for example, HAV, are able to grow without inflicting major damage to the cultured host cells. Moreover, even typically cytocidal viruses, such as poliovirus and some other, are able to establish, in certain cells or under certain conditions, persistent infection not accompanied with overt CPE [1] , although it should be noted that viral reproduction in persistently infected cells is usually not as efficient as in productively infected ones.
Picornaviruses possess small ca. The leader protein L is encoded by only certain picornaviruses. L and 2A proteins of picornaviruses may or may not exhibit proteases activities and in the former case they are referred to as L pro and 2A pro , respectively.
Regardless of this difference, both proteins perform largely antidefensive functions and are called security proteins [2]. After accumulation of a certain level of viral proteins, translation of viral RNA is switched to its exponential replication.
The newly synthesized RNA molecules are also translated, and when sufficient amounts of capsid proteins are accumulated, they are assembled with genomic RNA molecules into virions. The viral progeny usually leave the cells owing to their lysis. Several types of the death of eukaryotic cells are now recognized [34] , among which the most common are apoptosis and necrosis.
Apoptosis results from implementation of a cell-encoded program aimed at elimination of cells unwanted for various reasons. Both mechanisms converge in a cascade of serine proteases caspases attacking essential cellular targets.
Until relatively recently, necrosis was considered to be passive fatal cellular damage due to exhaustion of cellular resources and destruction of intracellular infrastructure caused by various extrinsic and intrinsic factors.
Typical features of necrotic cells include swelling, increased permeability of the plasma membrane with its ultimate rupture, nuclear pyknosis. More recently, the existence of host-encoded necrotic programs is well established, which may also be initiated from the plasma membrane receptors or in response to intracellular disturbances, and may have different underlying mechanisms.
Both apoptotic and necrotic cell-encoded pathways may be controlled by the same upstream elements and may compete with each other 34 , Pathogen-induced necrotic death is usually accompanied with emitting various proinflammatory signals.
In addition to apoptosis and necrosis, at least two other cell death mechanisms were reported to be implemented in viral infection. Cells dying of pyroptosis a program involving activation of proinflammatory caspase-1 exhibit certain features of both apoptosis and necrosis, for example, permeabilization of the plasma membrane and DNA fragmentation. Autophagy, usually a prosurvival cell reaction, which is characterized by the formation of multiple double-membrane vesicles autophagosomes delivering their content to lysosomes for degradation, may be exploited by a virus for its reproduction, but in certain cases may result in death of infected cells.
Partial virus disarmament may be achieved by inactivating their security proteins, a set of proteins specifically dedicated to antidefensive functions [2]. Inactivation of security proteins does not kill viruses but usually decreases their reproductive potential. The latter effect is probably due to decreased viral resistance to the cellular defenses, because viruses with impaired security proteins exhibit milder deficiency in hosts with compromised innate immunity reviewed in [2]. Experiments with mutual disarmament of mengovirus MV , a strain of encephalomyocarditis virus EMCV, a cardiovirus , and its host HeLa cells were particularly informative.
Infection with wild type wt virus terminated in necrosis, whereas MV mutants with inactivated L induced apoptosis [3]. However, in L — mutant-infected cells, a chemical inhibitor of apoptosis prevented not only apoptosis but also suppressed manifestations of major signs of necrotic CPE or delayed them until well after the completion of the viral reproduction [4].
The yield and time course of the reproduction of L — mutants were unaffected by this inhibitor. Thus, an adequate level of reproduction of a lytic virus can be maintained without immediate killing or even severely damaging its host cell, indicating that cells have enough resources to fulfill the needs of the virus and retain their own viability. Admittedly, the harvest of the L — mutants was somewhat lower compared with that of its wt counterpart.
However, the prolonged survival of L — -infected cells with pharmacologically switched-off apoptosis was not due to this lowered reproduction, because an MV with mutated RNA-dependent RNA polymerase generated the same amount of progeny but induced necrotic CPE with a wt-like time course. Thus, the major injures of MV-infected HeLa cells come from the fight between host defenses and viral counter-defenses rather than from the bold expropriation of cellular property by the virus.
The uncoupling viral reproduction and cellular pathology is not unique to the MV—HeLa system. Some lag between the CPE appearance and the peak of picornaviral reproduction under certain experimental conditions had been previously noted by several investigators 5 , 6 , 7 but this observation did not attract much attention.
The ability of picornaviruses to activate cellular apoptotic pathways was first discovered in poliovirus [8] and then described for numerous representatives of this viral family. The virus-triggered apoptosis appears to be an optional innate immunity reaction suppressing reproduction and spread of the pathogen.
Ample literature demonstrates that viral infections may change the balance between proapoptotic and antiapoptotic host factors. Alterations in favor of proapoptotic proteins are sufficient to elicit suicidal reaction [9]. Such a switch can be caused by activation of unspecific innate immunity mechanisms through sensors of viral infection as well as by interaction of viral proteins with components of the host apoptotic pathways.
Although several RNA- and DNA-containing viruses trigger apoptosis from without, only a few such examples are reported for picornaviruses 10 , Although other examples of the dependence of the apoptosis-triggering capacity of picornaviruses on the properties of their capsid proteins are known [12] , it is unclear whether this dependence is linked to the activation of the extrinsic apoptotic pathway or to the viral competence to efficiently infect the cells.
In most known cases apoptotic response is caused by replicating picornaviruses, although modulations of the apoptotic program through poliovirus interaction with its receptor was documented [13]. To suppress the defensive apoptotic reaction, viruses have evolved antiapoptotic tools.
Indeed, cells infected with poliovirus 8 , 14 and coxsackievirus [15] become resistant to nonviral apoptosis inducers. The dominance of antiapoptotic factors can be achieved by either downregulation of cellular proapoptotic activities or upregulation of antiapoptotic ones.
Picornaviruses may not only activate the intrinsic antiapoptotic machinery thereby maintaining acceptable conditions for growth in the host cell but also enhance resistance to death receptor-dependent apoptosis. Another variant of death of virus-infected cells is necrosis. This type of CPE is usually ascribed to the host unspecific exhaustion and damage caused by the lost competition with the virus for resources and infrastructure. Infection with many picornaviruses leads to inhibition of host translation and transcription, increased plasma membrane permeability, ionic disbalance, and damaged intracellular traffic [17].
However, to what extent these alterations are responsible for the usually rapid death of the infected cells is questionable.
Important contributions to the inhibition of host translation and transcription are made by picornavirus proteases 2A pro and 3C pro , which may target appropriate regulatory factors 18 , However, even such picornaviruses as cardioviruses, 2A proteins of which are devoid of protease activity may nevertheless suppress host translation [20] and transcription [21] by exploiting capacities of these nonenzymatic proteins to interact with host components 20 , 22 , Viral proteins may also cause other types of injuries.
Thus, infection with both enteroviruses [24] and cardioviruses [25] leads to enhanced permeability of the nuclear envelope but although this effect is due to proteolysis of nucleoporins by the viral 2A pro of enteroviruses 26 , 27 , 28 , cardioviruses elicit phosphorylation of nucleoporins triggered by the L protein 29 , Viral hydrophobic nonenzymatic proteins 2B and 3A are important players in the alteration of the cellular membranes [31].
Links between nonenzymatic viral proteins and cellular injuries hint that these injuries may result from modifications of certain cellular pathways. We have recently proposed that not only apoptosis but also virus-triggered necrosis may represent manifestations of host-encoded death programs, bona fide members of the innate immunity system [4]. A strong argument for this hypothesis is provided by a striking multifunctionality of the small 67 amino acids nonenzymatic cardiovirus L protein.
If apoptosis of infected HeLa cells was pharmacologically suppressed, diverse signs of necrotic CPE such as permeabilization of the plasma membrane, rearrangements of microtubule and microfilament networks, changes in the cellular and nuclear shapes, condensation of chromatin, and loss of the general metabolic activity all depend on L functionality [4]. This protein also impairs cellular interferon system [32] , formation of stress granules [33] , and, as mentioned above, nucleocytoplasmic traffic [25].
In addition, it exhibits antiapoptotic activity [3]. Admittedly, one cannot rigorously exclude the possibility that L directly affects such a multitude of targets by itself but it is much more likely that it modulates the activity of a single or few cellular control element s.
We hypothesize that this putative element is a part of the innate immunity system involved in deciding the fate of the virus-infected cell. Competitive apoptotic and necrotic pathways controlled by a shared upstream element are exemplified by the Ripoptosome, a complex involving protein kinases RIP1 and RIP3 35 , 36 , Rather some unknown, RIP3-independent necrotic pathway should operate in this system.
The proposed hypothesis on the existence of host-encoded virus-triggered necrotic pathway s by no means negates major effects of viral functions on this pathway.
A great variability of morphological and biochemical manifestations of necrotic CPE caused by different picornaviruses unambiguously indicates viral contributions to these manifestations. Therefore, new more efficient methods of drug discovery are needed to identify novel antiviral compounds from previously unmined libraries 2.
Because many medically important viruses use the lytic life cycle, drug discovery assays that measure host cell health can inversely report viral activity 3.
Traditionally, time-consuming, onerous, morphology-based plaque assays have been used to score cytopathic effect CPE. However, these assays are poorly suited for high-throughput screening HTS due to poor sensitivity or multiple assay steps or both.
We have developed a bioluminescent assay that measures cellular ATP, a tightly regulated molecule in cells, which serves as an excellent surrogate for host cell viability. Furthermore, the assay could also be easily configured to monitor vaccine development trials by measuring the serum neutralizing potential or immune cell responses directed at the viral target from vaccinated subjects or both.
Understanding and optimizing host cell health and viral infectivity parameters are critical steps for any successful in vitro cytolytic virology effort 4. Viral infectivity, for instance, is greatly affected by host cell receptor expression and distribution. Efforts to define a susceptible host cell called viral tropism and optimal conditions for susceptibility often linked to cell cycle, passage level and culture conditions can improve CPE and assay results.
Experimentally, this is addressed by titrating the host cell in the presence of a predetermined excess of virus. Titrating host cells without virus is necessary to establish the linear response range of the ATP detection assay.
An exposure period that is too short may lead to suboptimal CPE. If the exposure is too long, it may adversely affect the health of uninfected or untreated control cells. Once infection models are optimized, relative infective virus titers can be determined. For this step, working pools of influenza A, VEEV, dengue virus, and RSV were serially diluted in half-log increments and applied to their respective host cells.
Medium only was applied to control host cell wells as a virus-free control. Depending upon the kinetics of CPE determined during model development , viral titrations were incubated for 72— hours. Luminescence was measured and correlated with viral dilutions. The dilutions containing infectious virus demonstrated low cell viability and therefore low luminescence values due to widespread CPE. Conversely, dilutions containing no or limiting virus displayed higher luminescence values.
The off-target set of wells received medium without virus to address dose-dependent agent effects. After the appropriate exposure periods, the ATP Detection Reagent was prepared and added to plates as described previously. Luminescence was measured and correlated to antiviral agent concentration for both viral on-target and medium off-target exposures.
Off-target toxicity data were plotted on the same graphs and fitted in the case where cytotoxicity was present ribavirin on BHK-1 cells. Thirty thousand compounds from the Enamine chemical diversity library were screened as single doses in well plates against three influenza A strains. Infected and uninfected control wells were arrayed on each plate to statistically assess assay robustness. After the cells were incubated for 72 hours with the library compounds, the ATP Detection Reagent was added to the well plates and luminescence measured.
It offers a highly sensitive, precise and quantitative method of measuring CPE with an easy and fast workflow, an advantage compared to alternate methods. The assay successfully established both TCID50 and on- and off-target potencies for known antiviral compounds against medically relevant viral targets. We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems.
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