key: cord-0700448-rsbgd1tg
authors: Dowgier, Giulia; Maier, Helena J.
title: Quantification of Coronaviruses by Titration In Vitro and Ex Vivo
date: 2020-05-11
journal: Coronaviruses
DOI: 10.1007/978-1-0716-0900-2_11
sha: 8878b7a607e1159404061041291bdce891c36706
doc_id: 700448
cord_uid: rsbgd1tg

Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).

With the new-generation technologies progressing at a fast pace, high-throughput techniques are becoming available straight onto the bench, providing fast and accurate methods for routine tests in the lab. Detection of virus particles or selected antigens and their quantification are made possible in a short time in an easy way, thus establishing new laboratory standards [1, 2] . Detection of viral particles based on physical properties is commonly performed using specialized techniques that combine advanced optics and microfluidics, such as flow cytometry [1, 3] , light scattering [4] , capillary electrophoresis [5] , or fluorescence correlation spectroscopy [6] , providing fast results with high sensitivity. However, questions regarding the biological properties of viral particles analyzed through physical detection methods or nucleic acid amplification techniques (NAAT) remain a conundrum when identifying infectious particles is key. Combining analysis of physical particles and biochemical properties may answer this question, but this is not widely applicable. Classic virological techniques are therefore still needed to quantify virus infectivity. These techniques exploit the fact that viruses propagate in biological systems, such as cell culture, embryonated eggs, or organ cultures, and replication is generally accompanied by morphological or functional changes dictated by the number of infectious particles [7] . In this chapter we provide protocols for the quantification of coronaviruses using different methods that are applicable depending on the sample, virus, and, inevitably, laboratory capability. The tissue culture infectious dose/ 50 (TCID 50 ) or plaque assay titrations are described in detail for coronaviruses that have been adapted to grow in cultures of primary cells or continuous cell lines, providing results respectively in tissue culture infectious dose/ 50 (TCID 50 ) or plaque-forming unit (PFU) per volume of sample. Additionally, we describe a titration method for avian infectious bronchitis virus (IBV) adapted from Cook et al. [8] , using trachea organ cultures (TOCs). This is applicable for viruses that cause ciliostasis, providing results in ciliostatic dose 50 (CD 50 ), and representing an alternative to titrations carried out in embryonated eggs (EID 50 ) for those viruses not adapted to cell culture, thus limiting the required number of animals in compliance with the 3R principle.

All these methods provide accurate titrations; however, they are not always applicable for every virus. Although propagation of a virus in cell culture is generally accompanied by changes in cell morphology (referred to as cytopathic effect or CPE), which can be visualized using a microscope, some viruses do not induce CPE. In addition, use of TOCs for titration of respiratory viruses relies on observation and scoring of the cilia beating, as viruses replicating within the epithelia generally cause ciliostasis. However, some virus strains may be poorly ciliostatic; therefore, other techniques need to be used in these cases to quantify the virus.

To assess viral titer, a sample containing virus is diluted tenfold or twofold, depending on the expected virus concentration, and is used to infect tissue cultures or TOCs. Several days later or during the course of the infection, the cytopathic effect or the ciliostasis is recorded. From these data, the virus titer is calculated using the methods described by Spearman and Kaerber [9, 10] or Reed and Muench [11] . Importantly, these calculations apply to mismatched group sizes, as may happen when TOCs are lost due to bacterial infections or aspecific death. The virus titer is defined as the reciprocal of the dilution at which 50% of the inoculated tissue cultures show CPE or at which 50% of the inoculated TOCs show no residual beating of cilia.

Current limitations in adopting cell-based techniques are related to strain specificity in terms of host range and tropism. As many field strains of coronaviruses do not grow in cell culture, this limits the application of some techniques in principle. However, most isolates can be adapted to propagate in vitro in primary cells or continuous cell lines upon serial passage, selecting for the fittest subpopulations, eventually acquiring mutations related to cell-culture adaptation and as a result altering virus characteristics in vivo [12] [13] [14] . Here we describe protocols for titrating IBV by plaque assay or CD 50 and porcine deltacoronavirus (PDCoV) by TCID 50 . These protocols can easily be adapted for use with different coronaviruses and different cells, depending on the culture conditions required for the virus in use. 1. Ninety-six-well plates containing 80-100% confluent LLC-PK1 cells.

2. Medium Àtrypsin: EMEM supplemented with 1% HEPES, 1% nonessential amino acids, and 1% antibiotic antimycotic.

3. Medium +trypsin: Medium Àtrypsin supplemented with 10 μg/ml trypsin (see Note 1).

5. Multichannel aspirator.

6. Multichannel pipette.

7. 37 C cell culture incubator with 5% CO 2 .

8. Inverted microscope. 6. Add 0.5 ml of 0.1% crystal violet to each well or the minimum volume to just cover each well.

7. Incubate at room temperature for 10 min.

8. Remove crystal violet and dispose of according to local regulations.

9. Wash the plate by shaking upside down in a sink of water.

10. Pat plate dry and leave upside down at room temperature to fully dry.

11. Plaques should be clearly visible as holes in the monolayer varying in size and morphology based on the IBV strain (Fig. 2) . Count the number of plaques per well at the dilution with clearly defined, individual (not over-lapping) plaques (typically 10-50 plaques/well). Ensure duplicate wells are counted and an average taken.

12. Determine titer using the following equation:

Titer PFU=ml ð Þ¼ average number of plaques dilution factor Á inoculum volume ml ð Þ 13. For the most accurate results, the plaque assay should be repeated three times and the average titer determined. 2. Remove media from the tubes and wash once with sterile PBS.

3. Remove PBS from the tubes and add 500 μl of diluted virus to each tube selected for that dilution (see Note 6).

4. Incubate the tubes at 37 C for 6 days before assessing the titer by scoring the ciliary activity under the light microscope. TOCs are scored positive for infection when cilia activity is completely abrogated with a tolerance of 5% cilia still beating, whereas negative when residual activity is recorded up to 95% (see Note 7).

Determine the titers using the Reed and Muench calculations looking at the log dilutions and TOCs scores as follows:

(Log dilution above 50%) + (proportionate distance  log dilution factor) ¼ log ID 50 where the proportionate distance is calculated as follows:

Proportionate distance ¼ %positive above 50 ð Þ À 50% positive above 50% À positive below 50% ð Þ

The log ID 50 represents the end-point dilution at which the 50% of the TOC score positive. The dilution factor is finally applied accordingly to what applied, generating the final log CD 50 /ml. 3. If the likely titer of the virus is not known, use tenfold serial dilutions to identify the best range. If required, subsequently using twofold serial dilutions can provide a more accurate titer.

4. Alternative methods also exist for mixing media and agar. If there is concern regarding the overlay setting too quickly or risk of contamination from the water bath, hot agar can be mixed directly with cold media (4 C). Once the mixture feels warm to the touch, rather than hot, it can be added to cells.

5. The simplest method for removing agar from the cells is to hold the plate upside down with the lid removed. The small spatula is inserted between the agar and the wall of the well. Once the base of the well is reached, a small amount of pressure is applied to remove the agar, being careful not to scrape off the cells. The whole agar plug should then fall out easily. 6 . A quick and easy way to speed up the washing step during TOC titration is to add a few milliliters of PBS without removing the media in the tube, then with a rapid and confident rotation of the hand, pour the media/PBS mix onto a stack of tissues being careful not to lose the TOC ring. Finally, remove the excess PBS by aspiration or using a pipette. This step speeds up the procedure taking into account that many tubes, often more than 100, may need to be processed.

7. Assessing cilia activity during TOC titration may seem a subjective interpretation; however, the main effect on the TOC lumen is in reality quite striking at 6 days postinfection for viruses that are ciliostatic, usually leaving no doubt about the results. However, if a virus is poorly ciliostatic, this test should not be the first choice for quantification unless virus is detected by other techniques, such as antibody-based assays or probebased techniques.

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