Journal of Virological Methods 130 (2005) 117–123

A novel and rapid method to quantify cytolytic replicationof picornaviruses in cell culture

Per Andersson, Stina Alm, Kjell Edman, A. Michael Lindberg∗

Department of Chemistry and Biomedical Sciences, University of Kalmar, Smalandsgatan 24, S-39182 Kalmar, Sweden

Received 24 March 2005; received in revised form 16 June 2005; accepted 23 June 2005Available online 15 August 2005

Abstract

Determining viral titers is a key issue in a wide variety of studies regarding different aspects of virology. The standard methods used fordetermining picornavirus titers are endpoint titration assay and plaque assay, both time consuming and laborious. The method described uses thetetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) that is reduced to formazane by cellular dehydrogenase,genes shown to be down-regulated during picornavirus infection. The amount formazane produced correlates with the viral titers obtainedand can easily be measured using an ELISA plate reader. The colorimetric method has been evaluated using virus types from different genera

ccuracy.

cellus

n-iso-

whene-llsla-

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ontionationt al.,ursnv-t forandthein

of thePicornaviridae family. The MTT method reduces the time spent on determining the viral titers and still maintains a reliable a© 2005 Elsevier B.V. All rights reserved.

Keywords: Picornavirus; Quantification; Viral titer; Endpoint titration; MTT

1. Introduction

The Picornaviridae family constitutes nonenvelopedviruses with a single stranded RNA genome of positivepolarity. The family contains several human and other ani-mal pathogens, causing a wide variety of diseases includ-ing aseptic meningitis, myocarditis, foot and mouth disease,poliomyelitis, as well as several other diseases (Pallansch andRoos, 2001).

Picornavirus, like all other animal viruses, initiate infec-tion by attaching to a specific receptor (Rossmann et al., 2002;Smith and Helenius, 2004). The RNA genome is uncoated(Hogle, 2002; Smith and Helenius, 2004), enters the cyto-plasm and is subsequently translated to provide viral proteinsnecessary for genome replication (Agol et al., 1999; Pel-letier and Sonenberg, 1989). The polyprotein synthesizedis then processed to form precursors and mature viral pro-teins. Several copies of positive strand viral RNA is generatedby reusing a limited number of complementary (negative)strands as templates (Bedard and Semler, 2004). Late in infec-

∗ Corresponding author. Tel.: +46 480 446240; fax: +46 480 446262.E-mail address: michael.lindberg@hik.se (A.M. Lindberg).

tion, new virions are assembled and released from the(Racaniello, 2001). The infectious cycle of a picornavirusually ends by destruction of the infected cell (Thompsonand Sarnow, 2000). However, hepatitis A virus and Ljugan virus are two types of picornaviruses where thelated wildtype strains do not cause extensive damagereplicating in cultured cells (Johansson et al., 2004; Tichurst et al., 1983). During picornavirus infection, the cemetabolism is redirected towards viral replication and trantion. In the case of some members of the picornavirus fae.g. enteroviruses and rhinorviruses, it has been showmainly the 2Apro and 3Cpro protein accomplishes this rediretion. The 2Apro by cleaving the cellular translation initiatifactor eIF4GI/II, leading to a loss of the cap-binding funcand thereby inhibiting the cap-dependent cellular transl(Etchison et al., 1982; Kuechler et al., 2002; Trachsel e1980). This translational host cell shut-off normally occ2 h post-infection (Gan et al., 1998). In addition, it has beeshown for polioviruses that the 3Cpro target and cleave seeral transcription factors and other proteins importantranscription and processing of eukaryotic class I, IIIII transcripts significantly reducing the transcription ofhost cell genes (Kuyumcu-Martinez et al., 2002; Rubinste

0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.jviromet.2005.06.016

118 P. Andersson et al. / Journal of Virological Methods 130 (2005) 117–123

and Dasgupta, 1989; Shen et al., 1996; Yalamanchili et al.,1996).

Apoptosis has been identified as an important host defensemechanism for eliminating infected cells as well as inter-fering with virus replication (Hay and Kannourakis, 2002).Although many viruses can induce apoptosis, some DNAviruses, such as poxvirus and adenovirus are capable of sup-pressing the apoptotic cell death by expressing antiapoptoticviral genes to prevent the cell death (Shen and Shenk, 1995).In contrast, several RNA viruses including members of thePicornaviridae employ a rapid replication and translation incombination with a lytic infection while both stimulating andpreventing different parts of the apoptosis cascade (Ahn et al.,2003; Belov et al., 2003; Goswami et al., 2004; Henke et al.,2001; Koyama et al., 2000; Liu et al., 2004; Luo et al., 2002;Peng et al., 2004). Poliovirus in cell culture has been shownto interfere directly with the apoptotic pathway in differentways, involving mitochondrial damage, cytochrome c effluxand consecutive activation of caspase-3 and caspase-9 (Belovet al., 2003). The poliovirus protease 2A is able to interactand degrade the procaspase-9 and thereby inhibit the apop-totic process (Belov et al., 2003).

It has been shown that infection of mouse myocardialtissue by picornaviruses lead to dramatic change in the mito-chondrial activity and the cell metabolism (Koundouris et al.,2000). It was also demonstrated that infection of coxsack-i riale ds(

is ani osto idelyu assay( 6V ssed ap ose5 eth-o quired

l-t pedf thatM nated el-l nr uc-ct ion.T fectswo for-m MTTd clud-i rials( ta r’s

disease (Soriano et al., 2003). The MTT dye has also beenused to study various aspects of viral effects on cells, forinstance determining influenza virus titers in a system wherethe viral infection does not cause a cytopathic effect (Levi etal., 1995), and also for the study of antiviral effects (de Jalonet al., 2003). It has also been shown that certain pharmaco-logical compound can alter the MTT to formazane reductionalone, for instance the DT-diaphorase greatly enhances theMTT reduction (Goodwin et al., 1996b).

A novel and rapid method for quantitation of picor-naviruses is described by measuring cell viability using theMTT colorimetric method. The experimental setup is com-pared with the TCID50 method and the results are completelyin accordance with titers obtained by the TCID50 method, butwith the advantage that the values corresponding to viral titersare obtained in 48 h instead of 7 days.

2. Materials and methods

2.1. Cell cultures

Continuous Green Monkey Kidney Cells (GMK-G, a localvariant) and human lung carcinoma (A549) were maintainedin DMEM (Sigma–Aldrich), containing 5% Fetal BovineSerum (FBS) (Biological Industries) at 37◦C in a humidi-fi

2

V-1,k ex,U -1 ofNC lL edr ),B ),r ApG tiono D-w ngL dedp

hiss11k as-t 4s taS ox-s )a l.,

evirus B3, leads to a down-regulation of the mitochondnzymes involved in�-oxidation of saturated fatty aciTaylor et al., 2000).

Measuring the amount of infectious units in a samplemportant tool in virology. The viral titer is determined mften using biological assays. The standard and most wsed methods are plaque assay and endpoint titrationDulbecco and Vogt, 1954; Hierholzer and Killington, 199).alues determined by these separate methods are exprelaque forming unit (PFU) and tissue culture infectious d0% (TCID50), respectively. A disadvantage with these mds in general is that they are time consuming and reaily attention for approximately 5–10 days.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyetrazolium-bromide), colorimetric method was develoor quantitation of cell viability and is based on the factTT is reduced to formazane by the mitochondrial succiehydrogenase (Mosmann, 1983) as well as several other c

ular dehydrogenase (Slater et al., 1963). It has been showecently that poliovirus induces early inhibition of the sinate dehydrogenase activity (Koundouris et al., 2000) andhereby leading to an inhibition in the cellular respirathis phenomenon was observed before any cytolytic efere detectable (Koundouris et al., 2000). The implicationf these data is that uninfected cells may reduce MTT toazane to a greater extent than the infected cells. Theye has been used previously for several purposes in

ng cytotoxicity of several substances, such as biomatePariente et al., 1998), human growth hormone (Goodwin el., 1996a) as well as drugs for the treatment of Alzheime

s

ed 7.5% CO2 environment.

.2. Viruses

Infectious clones of Human Parechovirus-1 (pHPEindly provided by Glyn Stanway, University of Essnited Kingdom (Nateri et al., 2000)), Mengovirus (pM16, kindly provided by Willem Melchers, Universityijmegen, The Netherlands (Duke and Palmenberg, 1989)),oxsackievirus B5 strainDalldorf (pCVB5D-wt, Michaeindberg, University of Kalmar, Sweden, unpublishesults), were linearized usingMluI (New England BiolabsamHI (MBI Fermentas) andNotI (New England Biolabs

espectively, followed by in vitro transcription using T7 RNolymerase included in the MEGAScript® kit (Ambion).eneration of replicating viruses were done by lipofecf the in vitro transcribed RNA in susceptible cells (CVB5t and M16-1 in GMK and HPEV-1 in A549 cells) usiipofectamine 2000 (Invitrogen) according to recommenrocedures by the manufacturer.

In addition, the following viruses were used in ttudy; Echovirus 7 strainWallace (EV7W, ATCC VR-047), Echovirus 15 strainCharleston (EV15C, ATCC VR-056), Coxsackievirus A21 strainKuykendall (CVA21KK,indly provided by Darren Shafren, University of Newcle, Australia (Newcombe et al., 2003)), Coxsackievirus A2trainHS4679 (CVA24HS4679, kindly provided by Agneamuelson, Huddinge University Hospital, Sweden), Cackievirus B6 strainSchmitt (CVB6S, ATCC VR-1037nd Ljungan virus strain87012 (LV87012 (Johansson et a

P. Andersson et al. / Journal of Virological Methods 130 (2005) 117–123 119

Table 1Showing the different viruses in thePicornaviridae family used in this study and their taxonomy according to the latest classification (van Regenmortel et al.,2001)

Virus Strain Abbreviation Species Genus

Coxsackievirus B5 Dalldorf CVB5D Human Enterovirus B EnterovirusCoxsackievirus B Schmidt CVB6S Human Enterovirus B EnterovirusEchovirus 7 Wallace EV7W Human Enterovirus B EnterovirusEchovirus 15 Charleston EV15C Human Enterovirus B EnterovirusCoxsackievirus A21 KuyKendall CVA21KK Human Enterovirus C EnterovirusCoxsackievirus A24 HS 4679 CVA24HS4679 Human Enterovirus C EnterovirusLjunganvirus 87012 LV87012 Ljungan virus ParechovirusHuman Parechovirus 1 Harris HPEV-1 Human Parechovirus ParechovirusEncephalomyocarditis virus Mengo M16-1 Encephalomyocarditis virus Cardiovirus

2004). All virus strains were propagated once in GMK cellsprior to use in this study (Table 1).

2.3. Endpoint titration assay

Endpoint titration assays were carried out twice on twodifferent occasions by two different individuals. The TCID50(endpoint titration assay) is described elsewhere (Dulbeccoand Vogt, 1954; Hierholzer and Killington, 1996). Brieflysusceptible cells were seeded in a 96 well micro titer plateat a density of approximately 1.6× 104 cells/well and cul-tured for 24 h in DMEM containing 5% FBS at 37◦C ina humidified 7.5% CO2 environment. The cells were thenrinsed twice with phosphate buffered saline (PBS) and virusdilutions (1:100–1:109) were added in a total volume of100�l/well. The cell-virus cultures were then maintainedat 37◦C in a humidified 7.5% CO2 environment in DMEM(Sigma–Aldrich) lacking FBS, for 7 days (total volume of200�l/well). The plates were read manually on a daily basisand signs of cytopathic effect were recorded. TCID50/mlvalues were calculated according to the Spearman Karbermethod (Dulbecco and Vogt, 1954; Hierholzer and Killing-ton, 1996).

2.4. The MTT assay

atesa -t BS.T ve, ina sd rilefi nt( 1t nt of4a /ml,a7 erer teda ISAp sw unin-

fected cells where the uninfected control cells were given thevalue of 1 (100%).

2.5. Determination of viral titers using the MTT assay

The relative absorbance values obtained were assembledinto a graph together with standard deviation (S.D.). TheTCID50/ml equivalent value was then calculated accordingto the Thompson method (Thompson, 1947) in conjunctionwith the Spearman Karber method (Dulbecco and Vogt, 1954;Hierholzer and Killington, 1996). Briefly, a cut off relativeabsorbance value was determined at a given time point cor-responding to a TCID50/ml value calculated in a standardendpoint titration assay. Positive cells (considered infected)were cell-containing wells with a relative absorbance valuebelow the cut off value. The mean proportional viral titerswere calculated by averaging the difference between the dilu-tion closest below the cut off value and the dilution just abovethe cut off value. The implications of this can be summed upin the following equation:

Viral titers (equivalent to TCID50/ml) = 10X

X = − (D − (5((C − P1) − (P2 − C)) + 0.5))

whereD is the dilution closest below the cut off value (C)and P1 is the relative absorbance value of dilutionD andP ovet nceb stanti ibedp tv an beo

2

ho-s rties( fe ationa o dif-f tiona

Susceptible cells were seeded in 96 well microtiter plt a density of approximately 1.6× 104 cells/well and cul

ured for 24 h and the cells were then rinsed twice with Phe viruses were diluted and added as described abototal volume of 200�l/well. MTT (Molecular Probes) wailuted in PBS to a final concentration of 5 mg/ml, and steltered through a 0.22�m sterile filter. At a defined time poifor the initial experiments the time point was varied fromo 7 days and for the remaining experiments a time poi8 h post-infection was used), 20�l of the MTT solution wasdded to each well to give a final concentration of 0.45 mgnd the cells were further incubated at 37◦C in a humidified.5% CO2 environment for additional 4 h. The media weplaced with 100% DMSO (Sigma–Aldrich) and incubat 37◦C for 5 min. The cell lysates were then read in an ELlate reader (SpectracountTM, Packard) at 540 nm. All valueere calculated as relative active cells compared to the

2 is the relative absorbance value of the dilution just abheC value. The constant 5 corrects for the scale differeetween relative absorbance and dilution. The 0.5 con

s due to the dilution of 1:2 of virus in the assay as descrreviously. This equation will give a TCID50/ml equivalenalue. A spreadsheet for calculations of these values cbtained from the authors.

.6. Experimental determination of the cut off value

Two different viruses (LV87012 and CVB6S) were cen according to described differences in growth propeJohansson et al., 2004; Martino et al., 1999). In the case oach virus used in this study, three separate endpoint titrssays were run as described previously above, on tw

erent occasions, resulting in a total of six endpoint titrassays per virus.

120 P. Andersson et al. / Journal of Virological Methods 130 (2005) 117–123

Fig. 1. Microscopic images of Green Monkey Kidney Cells infected withEchovirus 15 strainCharleston and stained with the MTT salt. (A) Typicaluninfected GMK cells can be seen, (B) uninfected GMK cells treated withthe MTT can be observed, (C) endpoint titration cells were infected withEV15C and observed for cytopathic effect and (D) on day 3, the cells werelabeled with MTT to visualize the effect of the substance as described in theSection2.

The MTT assay was carried out three times per virus andthe time point when the MTT was added to the infectionsvaried from 1 to 7 days. In order to calculate the most reliableC value, the value was varied from 50 to 80% of the relativeactive cells in the equation described above.

3. Results

3.1. Determination of the cut off value

We were interested to evaluate a cell metabolic labelingmethod using MTT for quantifying cytolytic replication ofpicornaviruses. The first step was to determine a reliable andreproducibleC (threshold or cut off value) value. TheC valueshould be chosen to minimize time but still maintain accuracycompared to the endpoint titration assay.

Serial 10-fold dilutions of CVB6S and LV87012, virusesthat give different kind of cytopathic replication in cul-tured cells, were prepared and used to infect GMK cells asdescribed in Section2. The cultures were incubated furtherfor 1–7 days, after which the MTT assay was employed todetermine cell activity. From the results of this experiment(Figs. 1 and 2), the following conclusions were drawn: (1)differences in the virus concentration reflect upon cell viabil-i ctst

rem

Fig. 2. The effect of virus concentration in the samples and the duration ofthe incubation, based on GMK cell survival, as determined by color intensityin the MTT assay. All values are relative to the uninfected control. Note thatthe unit for the different bars indexed in the legend is Log virus dilutionranging from 0 to 9.

relative cell activity) on day 2 post-infection will yield aTCID50/ml estimate of 5.33± 0.03 (CVB6S) and 5.47± 0.03(LV87012) both corresponding to the endpoint titration assayobtained by titration of the viruses using standard methods(CVB6S: 5.38± 0.14, LV87012: 5.58± 0.20). AC value of0.5 (50% relative cell activity) on day 7 will give the expectedTCID50/ml (CVB6S: 5.40± 0.10, LV87012: 5.72± 0.03 cor-responding to the endpoint titration assay. As shown inFig. 3,the different calculated TCID50/ml can be observed at a dailybasis following different experiments using both the MTTassay and the endpoint titration assay.

3.2. Comparison of the MTT assay with the manual readendpoint titration assay

Using the cut off value determined (C = 0.8), the titers ofdifferent virus strains belonging to different picornavirus gen-era (Table 1) were analyzed both by manual endpoint titrationassays as well as the equivalent values in the MTT assays.The observed correlation between the obtained viral titers andthe corresponding MTT values are shown inFig. 4. Evaluat-ing the results obtained using the student’st-test showed thatthere is no significant difference between the endpoint titra-tion assay and the MTT assay (p value: >0.99). The resultsthus confirm that the use of the MTT method can, in a reliable

ty, (2) the length of the incubation prior to MTT assay affehe sensitivity of the assay.

To determine theC value, a series of calculations weade (data not shown) resulting in aC value of 0.8 (80%

P. Andersson et al. / Journal of Virological Methods 130 (2005) 117–123 121

Fig. 3. TCID50/ml equivalent values calculated using the differentC values 0.8 and 0.5 as well as the corresponding titer manually determined at different timepoints,n = 3. Using aC value of 0.8 on day 2 post-infection gives a corresponding value compared to the value calculated based on the endpoint titration assay.Error bars are given as standard deviation.

way replace traditional measurement of viral titers using stan-dard virological methods.

4. Discussion

The identification of viral infections in clinical specimensare done mainly by polymerase chain reaction (PCR) usingdefined sets of oligonucleotides capable of amplifying allknown pathogens. There is, however, still a need for isolat-ing and determining viral titers for studies on how a viralstrain interacts with a particular cell type. Accurate measure-ments of virus titers are, therefore, necessary for studies ofpathogenicity, virulence, evolution and environmental adap-tation of viruses.

Determining viral titers are thus of importance in manyvirological studies and the standard methods used are oftentime consuming and laborious. A common method to mea-sure viral titers used is the endpoint titration assay, wherecells are cultured in a 96 well microplate and infectedwith virus at different dilutions. The number of infectiousunits expressed as TCID50/ml can be calculated in approxi-mately 7 days post-infection. One of the major disadvantagesof the endpoint titration assay is that the assay is usu-

ally done manually, which increases the risk of erroneousresults.

The method described addresses this problem. The assayis rapid and reduces the time required from 7 to 2 days.More importantly, there is no manual assessment if cyto-pathic effect is observed or not, instead the activity of thecellular dehydrogenase is measured, genes shown to be down-regulated during picornavirus infection (Taylor et al., 2000;Wen et al., 2003). The method uses the tetrazolium salt MTTthat is reduced to formazane by cellular dehydrogenase. Theresult is then analyzed by measuring optical density (OD) at540 nm in an ELISA plate reader and comparing the valueobtained of infected and uninfected cells.

The initial step in this experiment was to determine theCvalue. TheC value is calculated as a determined maintainedcellular activity compared to uninfected cells at a minimumof time post-infection. Using theC value at a given timepoint should result in a TCID50/ml corresponding to the valueobtained from a series of endpoint titration assays. In thisassay, aC value of 0.8 (80% cellular activity maintained), onday 2 post-infection resulted in a titer corresponding to thetiter determined by the endpoint titration assay (Fig. 3) forviruses representing two genera of thePicornaviridae.

F eachw descr . TheT s des

ig. 4. Comparison of the MTT- and the endpoint titration assay. Forere run. The MTT assay was performed on day 2 post-infection asCID50/ml value calculated according to the endpoint titration assay a

virus, a number of endpoint titration assays (n = 4) as well as MTT assays (n = 4)ibed in the Section2. Error bars displaying standard deviation are also showncribed previously on day 7 post-infection.

122 P. Andersson et al. / Journal of Virological Methods 130 (2005) 117–123

TheC values were used later in a series of titrations andcalculations using several different human picornaviruses andwere found to correspond to the endpoint titration assaysobtained manually (Fig. 4). A statistical analysis (Student’st-test) showed that there is no significant difference betweenthe two groups of data.

In conclusion, using the colorimetric method described,based on the tetrazolium salt, MTT, reliable values wereobtained that correspond, completely to picornavirus titersgenerated by the TCID50 method. The time used for theanalysis is reduced by 75% and yet still maintains accuracycompared to the standard methods used at present.

Acknowledgements

We wish to thank Glyn Stanway for providing the pHPEV-1 clone, Willem Melchers for the pM16-1 clone, DarrenShafren for CVA21KK and Agneta Samuelson for providingCVA24HS4679. We would also like to thank Maria Gull-berg and Monika Somberg for their technical assistance. Thiswork was supported by grants from University of Kalmar, theKnowledge Foundation and the Research School for Pharma-ceutical Science, Sweden.

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