Which Of The Following Is An Unsuitable Culture Method For An Animal Virus?

Learning Objectives

By the stop of this section, you will be able to:

• Discuss why viruses were originally described as filterable agents
• Describe the tillage of viruses and specimen collection and handling
• Compare in vivo and in vitro techniques used to cultivate viruses

At the beginning of this affiliate, we described how porcelain Chamberland filters with pores pocket-sized plenty to permit viruses to pass through were used to discover TMV. Today, porcelain filters have been replaced with membrane filters and other devices used to isolate and identify viruses.

Isolation of Viruses

Dissimilar bacteria, many of which tin be grown on an bogus nutrient medium, viruses require a living host prison cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, so the growth medium can be harvested as a source of virus. Virions in the liquid medium can exist separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can and then be collected in the filtrate (meet Figure 6.sixteen).

Figure 6.16 Membrane filters tin be used to remove cells or viruses from a solution. (a) This scanning electron micrograph shows rod-shaped bacterial cells captured on the surface of a membrane filter. Note differences in the comparative size of the membrane pores and bacteria. Viruses will laissez passer through this filter. (b) The size of the pores in the filter determines what is captured on the surface of the filter (animal [cherry] and leaner [blueish]) and removed from liquid passing through. Annotation the viruses (green) pass through the finer filter. (credit a: modification of work past U.Southward. Department of Free energy)

Bank check Your Understanding

• What size filter pore is needed to collect a virus?

Cultivation of Viruses

Viruses can exist grown in vivo (within a whole living organism, plant, or creature) or in vitro (outside a living organism in cells in an artificial environment. Flat horizontal cell civilization flasks (encounter Figure six.17(a)) are a common vessel used for in vitro work. Bacteriophages can be grown in the presence of a dense layer of bacteria (as well called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (see Figure 6.17(b)). As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.

Figure 6.17 (a) Flasks like this may be used to civilization human or beast cells for viral culturing. (b) These plates contain bacteriophage T4 grown on an Escherichia coli lawn. Clear plaques are visible where host bacterial cells accept been lysed. Viral titers increase on the plates to the left. (credit a: modification of work by National Institutes of Health; credit b: modification of work by American Society for Microbiology)

Animal viruses crave cells within a host animal or tissue-civilization cells derived from an fauna. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, ii) production of vaccines, and 3) bones research studies. In vivo host sources can be a developing embryo in an embryonated bird's egg (e.g., craven, turkey) or a whole fauna. For example, well-nigh of the flu vaccine manufactured for almanac flu vaccination programs is cultured in hens' eggs.

The embryo or host animate being serves as an incubator for viral replication (meet Figure 6.18). Location within the embryo or host animal is important. Many viruses take a tissue tropism, and must therefore be introduced into a specific site for growth. Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may harm tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.

Figure 6.eighteen (a) The cells inside craven eggs are used to culture different types of viruses. (b) Viruses tin can be replicated in various locations inside the egg, including the chorioallantoic membrane, the amniotic crenel, and the yolk sac. (credit a: modification of work past "Chung Hoang"/YouTube)

For in vitro studies, various types of cells can be used to back up the growth of viruses. A primary prison cell culture is freshly prepared from animal organs or tissues. Cells are extracted from tissues by mechanical scraping or mincing to release cells or by an enzymatic method using trypsin or collagenase to break upwardly tissue and release single cells into intermission. Because of anchorage-dependence requirements, primary jail cell cultures require a liquid culture medium in a Petri dish or tissue-civilisation flask so cells have a solid surface such equally glass or plastic for zipper and growth. Primary cultures normally have a limited life span. When cells in a primary culture undergo mitosis and a sufficient density of cells is produced, cells come in contact with other cells. When this cell-to-cell-contact occurs, mitosis is triggered to terminate. This is called contact inhibition and information technology prevents the density of the cells from condign too high. To forbid contact inhibition, cells from the main prison cell culture must be transferred to another vessel with fresh growth medium. This is called a secondary cell culture. Periodically, jail cell density must be reduced by pouring off some cells and adding fresh medium to provide space and nutrients to maintain cell growth. In contrast to master jail cell cultures, continuous cell lines, ordinarily derived from transformed cells or tumors, are often able to exist subcultured many times or even grown indefinitely (in which instance they are called immortal). Continuous cell lines may not exhibit anchorage dependency (they will grow in break) and may take lost their contact inhibition. As a result, continuous cell lines tin grow in piles or lumps resembling small tumor growths (see Effigy half dozen.nineteen).

Figure 6.nineteen Cells for culture are prepared by separating them from their tissue matrix. (a) Master cell cultures abound attached to the surface of the culture container. Contact inhibition slows the growth of the cells once they go also dense and brainstorm touching each other. At this point, growth can simply exist sustained by making a secondary civilisation. (b) Continuous cell cultures are not affected by contact inhibition. They continue to grow regardless of cell density. (credit "micrographs": modification of work by Centers for Illness Control and Prevention)

An example of an immortal cell line is the HeLa prison cell line, which was originally cultivated from tumor cells obtained from Henrietta Lacks, a patient who died of cervical cancer in 1951. HeLa cells were the first continuous tissue-culture cell line and were used to establish tissue culture equally an important applied science for research in cell biology, virology, and medicine. Prior to the discovery of HeLa cells, scientists were not able to found tissue cultures with whatever reliability or stability. More than than six decades after, this cell line is still alive and being used for medical inquiry. Meet Eye on Ethics: The Immortal Jail cell Line of Henrietta Lacks to read more than almost this important cell line and the controversial ways by which it was obtained.

Check Your Understanding

• What property of cells makes periodic dilutions of primary cell cultures necessary?

Eye on Ideals

The Immortal Cell Line of Henrietta Lacks

In January 1951, Henrietta Lacks, a 30-year-old African American adult female from Baltimore, was diagnosed with cervical cancer at Johns Hopkins Hospital. We now know her cancer was acquired past the man papillomavirus (HPV). Cytopathic effects of the virus altered the characteristics of her cells in a process called transformation, which gives the cells the ability to split up continuously. This ability, of course, resulted in a malignant tumor that eventually killed Mrs. Lacks in October at age 31. Before her death, samples of her cancerous cells were taken without her knowledge or permission. The samples somewhen ended upwards in the possession of Dr. George Gey, a biomedical researcher at Johns Hopkins University. Gey was able to grow some of the cells from Lacks's sample, creating what is known today as the immortal HeLa prison cell line. These cells have the ability to alive and grow indefinitely and, even today, are still widely used in many areas of research.

Co-ordinate to Lacks's husband, neither Henrietta nor the family unit gave the hospital permission to collect her tissue specimen. Indeed, the family was not enlightened until 20 years after Lacks'south death that her cells were still live and actively being used for commercial and research purposes. Yet HeLa cells have been pivotal in numerous research discoveries related to polio, cancer, and AIDS, among other diseases. The cells have also been commercialized, although they accept never themselves been patented. Despite this, Henrietta Lacks's estate has never benefited from the employ of the cells, although, in 2013, the Lacks family was given control over the publication of the genetic sequence of her cells.

This case raises several bioethical problems surrounding patients' informed consent and the correct to know. At the time Lacks'south tissues were taken, at that place were no laws or guidelines about informed consent. Does that mean she was treated fairly at the time? Certainly by today's standards, the answer would be no. Harvesting tissue or organs from a dying patient without consent is not just considered unethical merely illegal, regardless of whether such an act could relieve other patients' lives. Is it upstanding, so, for scientists to keep to use Lacks's tissues for research, even though they were obtained illegally by today's standards?

Upstanding or not, Lacks's cells are widely used today for so many applications that information technology is impossible to listing them all. Is this a case in which the ends justify the means? Would Lacks exist pleased to know about her contribution to scientific discipline and the millions of people who have benefited? Would she want her family unit to exist compensated for the commercial products that have been developed using her cells? Or would she feel violated and exploited by the researchers who took part of her trunk without her consent? Considering she was never asked, we will never know.

Effigy 6.xx A multiphoton fluorescence image of HeLa cells in culture. Various fluorescent stains have been used to show the DNA (cyan), microtubules (dark-green), and Golgi apparatus (orange). (credit: modification of work by National Institutes of Health)

Detection of a Virus

Regardless of the method of cultivation, once a virus has been introduced into a whole host organism, embryo, or tissue-civilisation jail cell, a sample can exist prepared from the infected host, embryo, or cell line for further analysis under a brightfield, electron, or fluorescent microscope. Cytopathic furnishings (CPEs) are singled-out appreciable cell abnormalities due to viral infection. CPEs tin can include loss of adherence to the surface of the container, changes in cell shape from apartment to round, shrinkage of the nucleus, vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and complete cell lysis (see Figure 6.21).

Further pathological changes include viral disruption of the host genome and altering normal cells into transformed cells, which are the types of cells associated with carcinomas and sarcomas. The type or severity of the CPE depends on the blazon of virus involved. Figure six.21 lists CPEs for specific viruses.

Figure half-dozen.21 (credit "micrographs": modification of piece of work by American Social club for Microbiology)

Link to Learning

Lookout this video to learn well-nigh the effects of viruses on cells.

Hemagglutination Assay

A serological assay is used to notice the presence of certain types of viruses in patient serum. Serum is the harbinger-colored liquid fraction of blood plasma from which clotting factors take been removed. Serum can be used in a direct analysis called a hemagglutination assay to detect specific types of viruses in the patient's sample. Hemagglutination is the agglutination (clumping) together of erythrocytes (blood-red claret cells). Many viruses produce surface proteins or spikes called hemagglutinins that can bind to receptors on the membranes of erythrocytes and cause the cells to agglutinate. Hemagglutination is observable without using the microscope, merely this method does not always differentiate between infectious and noninfectious viral particles, since both can agglutinate erythrocytes.

To place a specific pathogenic virus using hemagglutination, we must employ an indirect approach. Proteins called antibodies, generated by the patient'due south allowed system to fight a specific virus, can exist used to bind to components such as hemagglutinins that are uniquely associated with specific types of viruses. The bounden of the antibodies with the hemagglutinins plant on the virus afterwards prevent erythrocytes from directly interacting with the virus. And then when erythrocytes are added to the antibody-coated viruses, there is no appearance of agglutination; agglutination has been inhibited. Nosotros call these types of indirect assays for virus-specific antibodies hemagglutination inhibition (HAI) assays. HAI can exist used to observe the presence of antibodies specific to many types of viruses that may be causing or have caused an infection in a patient even months or years later infection (see Figure 6.22). This assay is described in greater detail in Agglutination Assays.

Figure half-dozen.22 This chart shows the possible outcomes of a hemagglutination examination. Row A: Erythrocytes do non bind together and volition sink to the bottom of the well plate; this becomes visible every bit a crimson dot in the eye of the well. Row B: Many viruses have hemagglutinins that causes agglutination of erythrocytes; the resulting hemagglutination forms a lattice structure that results in red color throughout the well. Row C: Virus-specific antibody, the viruses, and the erythrocytes are added to the well plate. The virus-specific antibodies inhibit agglutination, as can be seen as a cherry-red dot in the bottom of the well. (credit: modification of work by Centers for Disease Command and Prevention)

Check Your Understanding

• What is the outcome of a positive HIA test?

Nucleic Acid Amplification Test

Nucleic acid amplification tests (NAAT) are used in molecular biology to detect unique nucleic acid sequences of viruses in patient samples. Polymerase concatenation reaction (PCR) is an NAAT used to observe the presence of viral Dna in a patient's tissue or torso fluid sample. PCR is a technique that amplifies (i.east., synthesizes many copies) of a viral DNA segment of involvement. Using PCR, short nucleotide sequences called primers bind to specific sequences of viral Dna, enabling identification of the virus.

Reverse transcriptase-PCR (RT-PCR) is an NAAT used to detect the presence of RNA viruses. RT-PCR differs from PCR in that the enzyme opposite transcriptase (RT) is used to make a cDNA from the small amount of viral RNA in the specimen. The cDNA can then exist amplified by PCR. Both PCR and RT-PCR are used to detect and confirm the presence of the viral nucleic acrid in patient specimens.

Case in Betoken

HPV Scare

Michelle, a 21-yr-old nursing student, came to the university clinic worried that she might accept been exposed to a sexually transmitted disease (STD). Her sexual partner had recently adult several bumps on the base of his penis. He had put off going to the md, but Michelle suspects they are genital warts caused by HPV. She is peculiarly concerned because she knows that HPV not only causes warts just is a prominent crusade of cervical cancer. She and her partner always use condoms for contraception, but she is not confident that this precaution volition protect her from HPV.

Michelle's physician finds no concrete signs of genital warts or any other STDs, but recommends that Michelle get a Pap smear along with an HPV test. The Pap smear will screen for abnormal cervical cells and the CPEs associated with HPV; the HPV exam will examination for the presence of the virus. If both tests are negative, Michelle can be more assured that she most likely has not become infected with HPV. Nevertheless, her doctor suggests it might be wise for Michelle to get vaccinated against HPV to protect herself from possible time to come exposure.

• Why does Michelle's physician order 2 different tests instead of relying on ane or the other?

Enzyme Immunoassay

Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and adhere to specific biomolecules called antigens. The detecting antibody attaches to the target antigen with a high caste of specificity in what might be a complex mixture of biomolecules. Also included in this type of analysis is a colorless enzyme attached to the detecting antibiotic. The enzyme acts as a tag on the detecting antibody and can interact with a colorless substrate, leading to the production of a colored end production. EIAs often rely on layers of antibodies to capture and react with antigens, all of which are attached to a membrane filter (come across Figure 6.23). EIAs for viral antigens are oftentimes used as preliminary screening tests. If the results are positive, further confirmation volition crave tests with even greater sensitivity, such as a western absorb or an NAAT. EIAs are discussed in more detail in EIAs and ELISAs.

Effigy 6.23 Similar to rapid, over-the-counter pregnancy tests, EIAs for viral antigens require a few drops of diluted patient serum or plasma applied to a membrane filter. The membrane filter has been previously modified and embedded with antibody to viral antigen and internal controls. Antibody cohabit is added to the filter, with the targeted antibiotic attached to the antigen (in the case of a positive test). Excess cohabit is washed off the filter. Substrate is added to activate the enzyme-mediated reaction to reveal the color change of a positive examination. (credit: modification of work by "Cavitri"/Wikimedia Eatables)

Cheque Your Agreement

• What typically indicates a positive EIA exam?

Clinical Focus

Part 3

Along with the RT/PCR assay, David'due south saliva was also collected for viral tillage. In full general, no single diagnostic exam is sufficient for antemortem diagnosis, since the results volition depend on the sensitivity of the assay, the quantity of virions present at the time of testing, and the timing of the assay, since release of virions in the saliva tin vary. As it turns out, the result was negative for viral cultivation from the saliva. This is not surprising to David's doctor, because one negative event is non an absolute indication of the absence of infection. Information technology may be that the number of virions in the saliva is low at the time of sampling. Information technology is not unusual to echo the examination at intervals to enhance the chance of detecting college virus loads.

• Should David'southward dr. change his course of treatment based on these test results?

Jump to the next Clinical Focus box. Get back to the previous Clinical Focus box.

Source: https://openstax.org/books/microbiology/pages/6-3-isolation-culture-and-identification-of-viruses

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