Indian Journ al of Experimcntal Biology Vol. 37, December 1999, pp. 1223-1 226

Comparison of two colorimetric assays to determine viral infectivity in micro culture virus titration

M M Parida, G Pandya, R Bhargava, R Bhattacharya" & A M Jana*

Di vis ions of Virology & "Toxico logy, Defence Research & Development Establishment , Gwalior 474002, 11l (1i ~1

Received 2 December 1998; revised 18 May 1999

Efficacy of two colorimetric assays, viz. MTT (3-4,5-dimethylthiazol-2-(yl-2,5-diphenyl tetrazo lium bro mide) and neutral red (NR) assays, performed by integrating the m to micro culture vi rus titrati on (MCVT), was comparcd with the conventional MCVT method in terms of percentages of infectivity and 50% infecti vity end points by employin g Polio viru s type-3 and Dengue virus type 4 as the cand idate viruses. The results suggested that MTT assay has an edge over NR assay as well as conventional MCVT method. For the first time, NR assay has been successfully empl oyed for the determinati on of virus infectivity titre.

Measurement of infec tivity pote ntial of a given virus is of paramount importance for accomplishing immu­nological studies, vaccine preparation etc. Cytopathic viruses have been conventionally quantitated by plaque assay and titrated for determination of 50 % infectivity end points . However, inherent problems e .g . manual counting of plaques apart from variation in plaque size and monitoring of cytopathic effects (CPE) for titration of infectivity render these tests vulnerable to erroneous results l

.

Colorimetric assays , on the other hand, quantify cell viability through enzyme mediated biochemical reactions owing to ingress of certain dyes inside the living cells . Mosmann2 and Borenfreund and Puemer' first advocated the application of tetrazolium (MIT) and neutral red (NR) assay respective ly, to quantitate cell viability and eventually the cytotoxicity to cells in vitro.

MTT assay, also known as te trazolium assay, has been exploited extensively to reveal the protective efficacy of therapeutic agents and plant extracts against cancer4

, human immuno deficiency virus (HIV -I) and herpes simplex viruss-7 and determina­tion of neutralizing antibody leve ls to HIV and respi­ratory syncytial virus8

,9. MIT assay using micro cul­ture virus titration (MCVT) was applied for the de­termination of infectivity titres of influenza viruses and was found to be compatible with the well estab­lished procedure of egg infectivity assay. Unlike MTT, neutral red dye uptake assay has not been sub-

*Correspondent author.

stantia]Jy exploited in virological researc h. R dye assay was earlier pe rformed for the study of anti viral efficacy of basil leaves ex tract against Polio virus t 3 (ref. 10) ype .

In the present study comparative efficacy of MTT and NR assay syste ms vis-a-vis conventi ona l method of determining infectivity titre e nd point in MCYT employing polio virus "type-3 (Poli o-3) dengue viru s type - 4 (DEN - 4) as candidate viruses have been re­ported.

Materials and Methods Cells-Human foetal lung epithelial ce ll line (L-

132) and African green monkey kidney ce ll line (Vero) obtained from National Centre for Cell Sci­ence (Pune, India) were maintained in our laborato ry by regular sub passaging us ing Eagle's minimum es­sential medium (EMEM) supple mented with 10% neonatal calf serum (NCS-Sigma, USA). L-132 and vero cell monolayers , be tween passage (P) leve ls 120-130 and P40-50 respective ly , were utili zed in the pre­sent study.

Candidate viruses-Polio-3 and DEN-4 viruses were obtained from National Institute of Health (Be­thesda, USA) and National Institute of Virology (Pune, India) respectively. They were maintain ed in our laboratory through serial passages , as pe r the standard procedure 1 I . Eventually both the viruses propagated at P 10 level were employed for the pres­ent study.

Antisera-Monospecific monkey antiserum against Polio-3 was obtained from Christian Medical College

1224 INDIAN J EXP BIOL. , DECEMBER 1999

(Ye llore, India) whereas anti serum to DEN-4 in mice was raised by us. These were subjected to checker boa rd titrati on in indirect ELISA before li se.

En zyme linked il11l11UnOsorbellt assay (ELISA)­Presence of the virus was confirmed by indirect ELISA based on the basic princip les described earlierl2 incorporating certain modi fications . The cell s were fixed with 10% phosphate buffe r saline (PBS) formalin on the attainment of 70-80% cytopathic effects (CPE), blocking the wells with 5% egg albumin (EA) , incubati ng at 37°C for 2 hrs and then addit ion of an tise rum of the respecti ve viruses . Goat anti human and rabbit anti mouse IgG horse fi:ld is h perox idase conjugates were ad led in the respecti ve ELISA plates . After in teraction wi th ortho phenylene diamine+H20 2. the reaction in the wells were stopped wi th 2N H2S04 and the optical densities recorded at 490 nm absorbance.

Conventional MC VT-Determinati on of ti ssue cu lture infecti ve dose 50% (TCIDso) end poim by conventi ona l method W :1S performed as per standard proced ure ll . Fres hl y tryps ini zed L- 132 eel! ,. contain­ing 5 x 104 cells/ml , suspended in EMEI\'! with 10% neonatal ca l f serum (NCS ) were seeded (100 pi/well ) in MT plate and incubated at 37°C in 5% CO2 ten­sion. Serial 10 fo ld dilUli ons of Poli o-3 and DEN-4 (10. 1 to 10'8) were prtpared alongs ide and 100 pI/well of each di lut j, ;n "vas addL'd in a set of 8 well s on to confluen t ce ll monolayer, ilrt1i~ rk ing two rows of healthy ce ll s (untreated) as contro l. Infected cell s were mOl1i t (' n~d each day for t h ~ development of viral induced CPE which occurI'd max imall y by day 3 post infection (PI) in casc of Polio-3 and 4 to 5 days PI in DEN-4. Ti s:-ue culture infec ti ve dose (TCID50)

was calculated according to the formula of Reed and Muench!.1.

Colorilllciric assays-MTT and NR dye ass <1ys pe rfo rmed in MCYT were identical in proceciura l deta ils up to the deve lopment of maxi mal CPE in­duced by d i rferent con-:ent rat ions of till' afo resaid Vil:uscS . Addi tional protocol s with regard to the two ~] ssay systems were as fo ll o'<'/s :

( i) MD' assay: MTT ass<lY for virus in fect ivity was performed in accorda n-:e with the method of Mos­rnanr? The ce il s we re wa:-.h ed with PBS on the maximal product ion of CPE and repleni shed with 100 pi/we ll of EM EM without phenol red . Tetrazoli um dye (Sign),], USA) dissolved in PBS (5mg/ml) was added in each well (10 pllwell) and incubated <1t 37°C in n CO2 incu bator for 3 hI'. Therea fter, 15 p i acid

propanol (0.04 N HCI in isopropanol) was added to

each well and mixed thoroughl y. Opti ca l dens iti es (00) were recorded at 570 nm abs I'bance ill an ELISA Reader (Dynatech, USA).

(ii) Neutral red (NR) assay: Thi s assay was ,IC­complished according to th ' procedure or Boren­freund and Puemer' with ce rtain modifi cat ions. Briefly , the ce ll s were was hed wit h P BS on the pro­duction of max imal CPE, fi xed with 4.5 % gluteralde­hyde solution, kept at roo m temperature for 45 mi n, further washed with PBS , stai ned with 0.2% NR dye aqueous solution and incll bated for I h I' at room tem­perature. After thorough washing [he sta ined cel ls were subjected to acid alcohol lys is by adding 100 p i of 0.5% aceti c ac id (Y IV) in 50% ethanol ro e; tch well. After 10 min of incubation, 00 values we re recorded as indicated abo ve.

Computation of TCID'i() in colorimefric O .I ,I'{fV.\'­

The infectivity end point (TCrD5(/1l11 ) of a given viru s was calculated by determining initi a ll y the cut ofT values. Cut off va lues were ca lcul ated according to Levi el al. l

• Ini tiall y the 00 va lucs of the blan ks compri sing mixtures used for the liberation of th e end products of the dyes (MTT and , IR) we re monit oreci and deduced from the ex perimental OD readings. Thereafter the cut off values were determined as fol­lows:

Mean OD value of untreated control \\'c ll s-2 X stan­dard devi ation (SO) Interpretation : OD < cut off va lue indicated

positivity OD > cut ofl va lue indicated nega­ti vity

Subsequentl y, the percentages of infecti vity f( lr each d il ut ion "vas calcula ted by t:lk in g propllrt ion 0[' p')Si ­tivity of 8 we ll s per diluti on. Fi nal ly, the 5fYir illfec ­tivity end point (log 10 TCID.'ic/Ill I) wa -; c,i1cuiakd ill accord ance with Reeel and MlIcncll forn ~ ul;ll '.

Results Co lorimetri c assays otTer mea~ I, Jr(': 1ll ' nt ori ent ed

results wh ic h have been desc ribed as tile mean of" minimum three repl icates. Ini tiall y th l' pl t'sencc nl

iruses IV ~IY ~ onri rmt:d by a 1 indircLI ELlS !\ whcrcir; fixed cell s exhibited p(l<-i tiVt' rcacriv it ) in infc-::cd well s as compared to re i:l tiVt' ncgat ive re~tcti \' il\' in contro l wells. An elevated 50;1, i ll(ccti \' it~ end puim of7 .0 (iog 10 TCID"ll i \\,~tS obs !'\'l:d ill MTT a :,~a:, ;t~

compared to 6.5 in NR and MC'YT a ~ .',:ty" in re~pect

PARIDA el al.: COMPARISON OF TWO COLORIMETRIC ASSAYS TO DETERMI NE VIRAL INFECTIV IT Y 1225

Table I-Comparative 50% infectivity end point (TCIDso) by colori metric assays (MTr and NR) and the conventional MCVT

Assay procedure

MTT NR MCVT

Polio-3 DEN-4

7.0 6.5 6.5

4.0 3.66 3.00

Log lI ) TCIDso was determined after calcul ati ng the proportions of posit ivity of 8 wells/dilution and thc cut off values thus obtaincd were designated Positive = > cut off value, Negati ve = < cut off value.

of Po1i0-3 (Table I). On the other hand, DENA ex­hibited rel ati vely reduced infectivity titres, though higher by MIT assay (4 .0) than NR and MCVT as­says which in turn revealed 3.66 and 3 .0 infectivity end po ints respecti ve ly. Poli o-3 vi rus revealed almost identical pe rcentages o f infectivity in conventional MCVT and NR assays except at 10.3 dilution of the virus w here in 100% infectivity was detected in con­trast to 87.5 % by MCVT (Fig. I). MIT assay in­variably revealed an edge over NR and MCVT. Even at the lowes t dilution of 10.8, 12.5% infecti vity was detected in MIT assay whereas no infecti viy was evident by the other two tests (Fig. I a). In DENA also the results indicated an invariably higher per­centages of infecti vi ty by MTT assay. A comparison between NR and MCVT revealed that NR exhibited e levated !nfectivity percentages as compared to MCVT (Fig. Ib).

The quantification of cell viability (expressed in terms of average of OD values) vis-a-vis correspond­ing virus dilutions of Poli o-3 and DEN-4 have been depicted in Fig. 2 (a and b) . A dose dependent re ­sponse is clearly evident as indicated by the increase in cell viability with proportionate decrease in the concentration of both the viru ses.

Discussion Tn colorime tric assays MTT and NR dyes get con­

ve rted into coloured produc ts in response to the bio­chemical reaction in the li ving cells. In turn these products can be measured in terms of optical densi­ti es. MTT-a Tetrazolium salt is a ye llow coloured dye (3-(4,5 dimethylthiazol 2-yl) 2,5 diphenyl tetrazolium bromide) which gets cleaved by mitochondrial succi­nate dehydrogenase enzy me into a blue co loE red for­mazan in active ce ll s. T his product does not form crysta ls when inte racted w ith isopropy l alcohol and

10)

q > ~ 10 QI

1: ~ MCVT ~ MTT ~ NR

H

Virus ditution

Fig. I-Comparative percentagcs of infcctivi ty in cOIl\"Cnt iOIl:l1 (MCVT), MTT and NR assays (a) Poli o-3, (11) DEN-4

10)

0.5

0.4

0.3

0.2

E 0.1 c a

0 .... '" ;; Ib)

ci 0.5 ci

0.4

0.3

0.2 o MTT t::,. NR

J L--~-..L'O-::'2-.J.'O-:·J;---"O-' 4;---', O~';;-s _ ' OL' 6'--_,Lo-" ---"0-'

0.1

0 10-1

Virus ditu tion

Fig. 2-Dose dependent respollsc of OD va l u c~ with corn.:spo nd · ing virus di lutions in MTT and NR assays. (al Pol io- 3, (b) DEN-4

thus can be accurately measured" . On the othe r hand , neutra l red-a weak cationic dye gets accumul ated in the Iysosomes of the li ving cells that can then be eX­

tracted by acid-a lc oho l Iys is·i. The conventional MCVT is a quanta l IJlethod wh ich

depends on the visua l observat ion and subjecti ve

1226 INDIAN J EXP BIOL. , DeCEMBER 1999

scoring of CPE, e licited in response to different concentration s of the viruses. Colorimetr ic assays on the contrary are independent of CPE manifestations , reveal the extent of ce ll viability quantitatively and thereby indirectly denote the virus infecti vity . MC VT when coupled with co lorimetric procedure ensures reproducibility, consistency and ease of pe rformance. Th is apart, in MIT assay there is no need to harvest the viabl e cells wherei n the ce ll viability can be di rectl y measured by a spectrophotomete r. In NR assay however, the ex traction of dye is an imperative step to record the correspond ing 00 values23 MIT dye has been reported to be applied in various assay systems where quantification of living cells can indirectl y denote the state of the cytotoxic cells viz. quantification of cel ls in myotoxic assay of snake

venomsl." assay of inte rl eukin-2 by determining he lper

T ce ll population!.', response of chemotherapy in l eukemia~ etc. NR dye was however, exploited only as an indicator of cytopathogenic ity of vi ruses in an interferon assayl 6. However, no direct reports on the application of NR dye for determining the virus infect ivity end point are availab le. Therefore to our knowledge this study may be the first attempt of titrating virus infecti vi ty by empl oy ing the NR dye.

In the present study, a titre of 7.0 TCrDsolml was detected in respect of Po li o<~ by MTT assay whereas a lower titre of 6.5 was ex hi b ited by both NR and MCVT assays. In comparison , DEN-4 showed relative ly decreased titres of 4.0, 3.6 and 3.0 TClD501ml by MCVT, N R and MCVT respectively (Table I ). Percentages of infectivity (Fi g. I ) further substantiated the relative efficacy of MIT assay to quantify the extent of CPE elicited by diffe rent dilutions of both the viruses. Dose dependent response as exhibited by colorimetric assays accurately measured the extent of cell damage by different concentrati ons of the two candidate viruses. The lower 00 values in MIT assay, as compared to NR assay at a given concentration of the virus, denote higher sensitivity of MIT which in tum represents the mitochondrial status of the cells (Fig. 2). Though a recent report suggested a good correlation between MIT and NR assays with regard to evaluation of cytotoxicityl 7, the present results

re vealed an edge of MTT assay system ove r both NR and the conventi onal MCVT in res pect o f in f'ect ivity end point detenninalion of the viruse.· .

The present study conclusive ly suggcs ts that th l: prec ision and ease of performance or colorimetr ic assays for the de te rminati on of thc infect ivi ty titres or cytopathic viruse ' o ffe r a better a lt e rnat i, 'c to thc

existing techn iques for infect ivity cie te rmination . Between the two co lorimetric a~ .. ay procedures. MTT is re lative ly more sens iti ve and less cumbersome thall NR assay.

Ackn()wledgement The authors are thankful to Dr R V Swamy,

Director, Defence Research and Deve lopment Establi shment , Gwalior and Shri K M Rao, Associate D irector and Head, Divi sion of Viro logy for the ir keen inte rest and encouragement in carrying ou t thi s work .

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