applied
sciences
A icle
Quan i a i e Flow Cy ome y o Measu e Vi al
P oduc ion Using In ec ious Panc ea ic Nec osis
Vi us as a Model: A P elimina y S udy
Diego Vázquez, Ca men López-Vázquez, JoséG. Ol ei a , Isabel Bandín
and Ca los P. Dopazo *
Ins i u o de Acuicul u a, Depa amen o de Mic obiología, Uni e sidad de San iago de Compos ela,
15782 San iago de Compos ela, Spain; [email p o ec ed] (D.V.); [email p o ec ed] (C.L.-V.);
[email p o ec ed] (J.G.O.); [email p o ec ed] (I.B.)
*Co espondence: [email p o ec ed]; Tel.: +34-8818-16048
Recei ed: 6 Sep embe 2018; Accep ed: 22 Sep embe 2018; Published: 26 Sep embe 2018
Abs ac :
In ecen decades, low cy ome y (FCM) has become an impo an ool in i ology, due o
i s applica ions in i al eplica ion and i al-cell in e ac ions, as well as i s capaci y o quan i y
p o eins (qFCM). In he p esen s udy, we ha e designed and e alua ed a qFCM p ocedu e o he
in i o
analysis and quan i ica ion o ish i al p o eins, using he in ec ious panc ea ic nec osis i us
(IPNV) as a model. We ha e also es ed i s use o i al i a ion and adap ed he MARIS (me hod o
analysing RNA ollowing in acellula so ing) me hod o simul aneous quan i ica ion o i al RNA
exp ession in in ec ed cells. The p ocedu e has p o ed o be epea able and ep oducible o an
accep able le el, al hough o ensu e ep oducibili y, he epe i ion o s anda d cu es is ine i able.
Rega ding i s use o i al quan i ica ion, a di ec ela ionship (by a second-deg ee polynomial
eg ession) be ween i al i es and Molecules o Equi alen Soluble Fluo och ome (MESF) was
obse ed. Finally, he esul s suppo he use o his echnology, no only o i us quan i ica ion,
bu also o s udy i al eplica ion om a quan i a i e app oach.
Keywo ds: qFCM; IPNV; i al eplica ion
1. In oduc ion
Since i s i s de elopmen , low cy ome y (FCM) has become a eliable ool o s udy i us-cell
in e ac ions and, among i s applica ions, he quan i ica ion o cellula an igens gained popula i y in
he ea ly 80s [
1
,
2
]. This me hodology, known as quan i a i e low cy ome y (qFCM), is de ined as
“ he calib a ed measu emen o luo escence in ensi y om labelled pa icles o de e mine he ac ual
numbe o luo escen ligands labelling each pa icle” [
3
]. Di e en clinical s udies using qFCM ha e
been published [
4
], including applica ions in i al esea ch, like moni o ing he mul iplici y o in ec ion
o a i us du ing accine p oduc ion, he e alua ion o p o ein he e ogenei y in he He pes simplex
i us, o he e alua ion o cell an i i al esponse o Rhino i us [5–7].
FCM has also been ex ensi ely used in ish i ology, o he sc eening o an ibody lib a ies [
8
],
o he de ec ion o di e en i uses such as IPNV [
9
–
13
], he Lymphocys is i us [
14
], he In ec ious
haema opoie ic nec osis i us (IHNV) [
15
] o he I ido i us [
16
], and in i al eplica ion s udies [
17
–
21
].
Howe e , o ou knowledge, i s use o eal quan i a i e pu poses has ne e been epo ed.
In ec ious panc ea ic nec osis is an impo an disease a ec ing salmonid aquacul u e. The causa i e
agen , he In ec ious panc ea ic nec osis i us (IPNV), is an Aquabi na i us, belonging o he amily
Bi na i idae. Membe s o his amily ha e a genome composed o wo segmen s o double-s anded
RNA (named A and B), and a naked icosahed al single-shelled capsid. Segmen A encodes a 105 KDa
Appl. Sci. 2018,8, 1734; doi:10.3390/app8101734 www.mdpi.com/jou nal/applsci
Appl. Sci. 2018,8, 1734 2 o 14
pVP2-NS-VP3 polyp o ein, which is co ansla ionally p ocessed and clea ed in o he s uc u al VP2
and VP3 i al p o eins, and he NS i al p o ease (also known as VP4) [
22
]. This segmen also
encodes a 17 KDa non-s uc u al p o ein called VP5 whose biological unc ion s ill needs o be
de e mined [
23
]. Segmen B encodes VP1, he i al RNA-dependen RNA-polyme ase (RdRp) [
24
].
Since VP2 elici s neu alizing an ibodies and ca ies se o ype-speci ic and g oup-speci ic an igenic
de e minan s, his p o ein is impo an o he de elopmen o subuni accines, o diagnosis and o
se ological yping.
Di e en applica ions o FCM ha e been employed o his i us, bo h o he de ec ion o he
i us in di e en cells and o
in i o
and
in i o
i al eplica ion s udies; al hough in some o hose
s udies FCM was used o de e mine he le el o p oduc ion o ce ain i al p o eins, a quan i a i e
app oach o his echnique has ye o be alida ed [17,20,25,26].
The objec i e o he p esen s udy has been he design and e alua ion o a qFCM p ocedu e o
he analysis and quan i ica ion o VP2 p o eins exp essed in
in i o
in ec ed bluegill y (BF-2) cells.
We ha e also adap ed he me hod o analysing RNA ollowing in acellula so ing (MARIS) epo ed
by H a in e al. [
27
] o he simul aneous quan i ica ion o he i al RNA exp ession in in ec ed cells.
2. Ma e ials and Me hods
2.1. Vi us and Cell Line Employed
Fo his s udy, he IPNV Wes Bux on (WB) s ain (ATCC VR-877) has been employed.
The BF-2 (Blue in gill) cell line (ECACC # 00021712) was used o i al p opaga ion. Cells we e
main ained a 20
◦
C in 25 cm
2
lasks wi h Eagle’s Minimum Essen ial Medium (EMEM, Gibco,
The mo ishe , Bilbao, Spain) supplemen ed wi h 10% oe al bo ine se um (FBS) (Lonza, Mad id,
Spain), penicillin (100 IU/mL) and s ep omycin (0.1 mg/mL) (Lonza, Mad id, Spain).
Fo i al p opaga ion and o he FCM assays desc ibed below, monolaye s wi h a ound 80%
con luence we e inocula ed wi h he i us a a mul iplici y o in ec ion (MOI) o 0.1–0.01 (o lowe ,
in some expe imen s). A e 1 h a 15
◦
C, he emaining inoculum was wi hd awn, and he monolaye s
co e ed wi h esh 2% EMEM (EMEM supplemen ed wi h 2% FBS and an ibio ics) and incuba ed a
15
◦
C ei he un il cy opa hic e ec (CPE) became ex ensi e o he p opaga ion o he i us, o du ing
he co esponding ime indica ed o each assay.
2.2. Ti a ion by Plaque and Endpoin Dilu ion Assays
Fo plaque assay i a ion, 6-well pla es wi h 80% con luen BF-2 monolaye we e employed.
Ten- old dilu ions o he i us we e inocula ed in he wells (3 eplicas pe dilu ion, 200
µ
L pe eplica)
and, a e 1 h o adso p ion a oom empe a u e, he emaining inoculum was emo ed, and he
monolaye s o e laid wi h 2 mL o 1.2% low mel ing aga ose (P onadisa, Mad id, Spain) in 2%-EMEM.
A e 5 days o incuba ion a 15
◦
C, he monolaye s we e ixed o 1 h wi h a ixa i e solu ion composed
o 25% o malin, 10% e hanol and 5% ace ic acid, in phospha e-bu e ed saline (PBS). A e ixa ion,
he aga ose o e lay was emo ed, and he monolaye s we e s ained wi h 1 mL o 5% c ys al iole
(Sigma, Mad id, Spain) in PBS. The wells we e washed, he plaques coun ed, and he i e exp essed as
plaque o ming uni s pe millili e (p u/mL).
Fo he endpoin dilu ion i a ion assay, 96-well mic o i e pla es wi h BF-2 cells in 2%-EMEM
we e employed. Ten- old dilu ions o he i us we e inocula ed in he wells (3 eplicas pe dilu ion,
100
µ
L pe eplica) and he pla es incuba ed a 15
◦
C o 1 week and examined o he p esence o CPE.
The i es we e de e mined as issue cul u e in ec ion dose pe millili e (TCID50/mL) [28].
2.3. MARIS S aining and Fluo escence Ac i a ed Cell So ing (FACS)
The MARIS s aining p ocedu e and FACS acquisi ion o posi i e in ec ed cells [
27
] was adap ed
and op imized o so IPNV in ec ed BF-2 cells, using a monoclonal an ibody agains he VP2 p o ein
and indi ec labelling wi h a FITC conjuga e. A e i al in ec ion in 25 cm
2
lasks, he cells we e
Appl. Sci. 2018,8, 1734 3 o 14
dispe sed using PBS and igo ous pipe ing o de ach hem om he lask. A e cen i uga ion a 500
×
g o 30 min, he supe na an was wi hd awn, and he cells we e ixed and pe meabilized o 30 min a
4
◦
C wi h 4% pa a o maldehyde (PFA, Pan eac, Ba celona, Spain) and 0.1% saponin (Sigma-Ald ich,
Mad id, Spain) solu ion in PBS supplemen ed wi h 1:100 RiboLock RNase inhibi o (The mo Scien i ic,
Bilbao, Spain). The cells we e hen cen i uged 5 min a 500
×
gand washed wice wi h a washing
bu e : PBS supplemen ed wi h 0.2% bo ine se um albumin (BSA), 0.1% saponin and 1:1000 RiboLock
RNase inhibi o . Incuba ion wi h p ima y monoclonal an ibody (an i-IPNV_VP2, IBT sys ems GmbH,
E inge , Ge many) was ca ied ou in s ain bu e (PBS, 1% BSA, 0.1% saponin and 1:100 RiboLock
RNase inhibi o ; Sigma-Ald ich, Mad id, Spain) du ing 60 min a oom empe a u e. The cells we e
washed wice in washing bu e ollowed by seconda y an ibody s aining (FITC conjuga ed goa
an i-mouse an ibody, Sigma-Ald ich, Mad id, Spain) o 45 min a oom empe a u e in s aining bu e .
The cells we e washed wice in wash bu e and esuspended in so bu e con aining 0.5% BSA
and 1:1000 RiboLock RNase inhibi o in PBS. The so ing p ocedu e was pe o med on a FACSA ia
(BD Biosciences, Mad id, Spain) using FACS Di a so wa e.
2.4. Quan i a i e Flow Cy ome y (qFCM) Assessmen
Fluo escence da a was expo ed o FlowJo Vx so wa e and analysed in e ms o o wa d ligh
sca e ing (FSC) and side-ligh sca e ing (SSC) o ga e cells om backg ound noise. Single e en s
we e disc imina ed om cell agg ega e ga ing e en s in plo s o o wa d-ligh sca e ing a ea (FSC-A)
agains o wa d-ligh sca e ing signal heigh (FSC-H). Fluo escence pa ame e s (in e ms o a bi a y
luo escence in ensi y; AFI) we e s anda dised in Molecules o Equi alen Soluble Fluo och ome
(MESF) uni s by means o ype IIIb s anda d mic osphe e beads [
29
] using a Quan umTM MESF Ki
(Bangs Labo a o ies, Inc., Bu ling on, ON, Canada). S anda d beads we e esuspended in he same
so bu e in o de o a oid pH-based di e ences be ween s anda ds and samples. The s anda d beads
we e analysed on he same day o he cell samples analysis, acco ding o he indica ions o he supplie .
The AFI geome ic mean o each bead popula ion was eco ded and he calib a ion cu e esul ing
om he linea eg ession o he geome ic mean channel numbe e sus he expec ed MESF alues o
he co esponding beads p o ided a slope o MESF pe luo escence channels using he QuickCal 2.3
(Bangs Labo a o ies, Inc., Bu ling on, ON, Canada). The Mean o Fluo escence In ensi y (MFI) was
hen calcula ed as MESF
×
pe cen age o in ec ed cells. Once he h eshold limi be ween posi i es
and nega i es was de e mined using nega i e con ols (mock in ec ed cells), posi i e cells (agains
IPNV VP2) we e so ed and collec ed in so bu e illed ubes.
2.5. RNA Isola ion
The collec ed VP2-IPNV posi i e cells we e pelle ed by cen i uga ion o 30 min a 500
×
gand
4
◦
C. The supe na an was disca ded and o al RNA isola ed using he ki Reco e All To al Nucleic
Acid Isola ion (Ambion, Mad id, Spain), ollowing he modi ied p o ocol desc ibed by H a in e al.,
(2014). The concen a ion o he ex ac ed RNA was quan i ied wi h a ND-1000 spec opho ome e
(Nanod op Technologies, Inc., Wilming on, CA, USA) and i s quali y was e alua ed om he a ios
A260/280 and A260/A230 as desc ibed by Samb ook e al. [30].
2.6. Quan i a i e RT-PCR
We ha e used a p e iously op imized e e se ansc ip ion-quan i a i e PCR (RT-qPCR) me hod
o he absolu e quan i ica ion o IPNV, using he RNA isola ed om so ed cells. The RNA was
ansc ibed o cDNA using Supe Sc ip III (In i ogen, Me elbeque, Belgium) o a 20
µ
L eac ion
olume. B ie ly: 9
µ
L o RNA we e mixed wi h 1
µ
L o andom p ime s (In i ogen, Me elbeque,
Belgium), hea ed o 99
◦
C o 5 min and immedia ely cooled down o 4
◦
C. Then, 4
µ
L (5
×
) o Fi s
S and Bu e , 1
µ
L di hio h ei ol (DTT) (0.1 M), 1
µ
L o dNTPs (10 mM), 3.8
µ
L H
2
O and 0.2
µ
L Supe
Sc ip III RT polyme ase we e added pe eac ion. The he mal p o ile con inued wi h 10 min a 25
◦
C,
50 min a 50
◦
C and 5 min a 85
◦
C. Finally, he cDNA was main ained a 4
◦
C un il use o s o ed a
Appl. Sci. 2018,8, 1734 4 o 14
−
20
◦
C. Real ime PCR ampli ica ions we e pe o med in a Bio-Rad CFX96 (Bio-Rad Labo a o ies, Inc.,
Mad id, Spain) using 10
µ
L o 2x SYBR
®
G een Supe mix (iQ SYBR
®
G een Supe mix, The mo ishe ,
Bilbao, Spain), 500 nM o each p ime (PP_WB 2370F, 5
0
-CAAGTTTGGCAGGCTCATCAG-3; PP_WB
2614R, 5
0
-CGTAGTCCTCGTACTCTTCTCC-3) and 2
µ
L o cDNA in a 20
µ
L inal eac ion, wi h he
ollowing he mal p o ile: 95
◦
C o 3 min, ollowed by 42 cycles o 95
◦
C o 15 s and 60
◦
C o 30 s, and a
mel ing cu e analysis o 55
◦
C o 95
◦
C wi h an inc emen o 0.2
◦
C o 10 s. Absolu e quan i ica ion
was pe o med using
in i o
ansc ibed RNA s anda d om a cloned plasmid wi h a 364 bp agmen
om RNA egion 2317–2681 o e e ence s ain WB [31].
2.7. P elimina y Op imiza ion o he Assay and E alua ion o qFCM Reliabili y
In a p elimina y app oach, se ial dilu ions o he i us we e inocula ed in BF-2 cells and, a e 24 h
incuba ion, p ocessed o qFCM as desc ibed abo e. A e he h eshold limi be ween posi i e and
nega i e e en s was es ablished using he mock in ec ed cells, posi i e e en s wi h di e en le els o
luo escence we e obse ed. The co ela ion be ween he MOI employed and he MFI was analysed by
means o linea eg ession using P ism V5.0 (G aphPad So wa e, Inc., La Jolla, CA, USA). In addi ion,
100,000 cells we e so ed o u he RNA isola ion and qPCR analysis, in o de o es he me hod o
eco e ing i al RNA om ixed BF2 posi i e cells desc ibed abo e.
2.8. Repea abili y and Rep oducibili y o Quan i a i e Flow Cy ome y Measu emen s
To assess he eliabili y o he qFCM in e ms o epea abili y and ep oducibili y o he luo escen
measu emen s, eplica e assays we e pe o med on h ee non-consecu i e days (3 simul aneous
eplicas pe day), using BF-2 in ec ed cells (a a MOI o 0.1) and he s anda d beads as desc ibed be o e.
A e wa ds, he luo escence da a in e ms o AFI, MESF and MFI we e subjec ed o analysis o he
coe icien o a ia ion (CV) alues.
2.9. Compa a i e E alua ion o Co ela ion be ween qFCM and T adi ional Ti a ion Me hods
Ten- old i al dilu ions we e inocula ed in 25 cm
2
lasks wi h semicon luen BF-2 cells (app oxima e
densi y 5
×
10
5
cells/cm
2
). Adso p ion was ca ied ou a oom empe a u e and, a e 60 min,
he inoculum was wi hd awn and eplaced by esh 2%-EMEM. In ec ed cul u es we e kep du ing 24 h
a 15
◦
C, and hen he cells om each lask we e collec ed and he luo escence pa ame e s quan i ied
as desc ibed abo e. The same i us sample was i a ed by wo di e en p ocedu es: plaque assay and
end poin dilu ion as desc ibed.
2.10. Assessmen o VP2 P o ein Exp ession du ing he IPNV Time Cou se In ec ion
Ten 25 cm
2
cul u ed lasks we e seeded wi h he same densi y o BF2 cells om a single 150 cm
2
con luen monolaye . When he subcul u ed monolaye s we e semicon luen , he i us was inocula ed
a a MOI o 0.1–0.01 and a e 1 h o adso p ion he monolaye s we e co e ed wi h 2%-EMEM and
incuba ed a 15
◦
C o a maximum o 24 h. The cells we e collec ed a he ollowing pos -in ec ion
(p.i.) imes: 0 h p.i. (collec ed igh a e adso p ion) and 4, 8, 12, 16, 20 and 24 h p.i. Mock in ec ed
cells (nega i e con ol o in ec ion) we e also collec ed a he end o he expe imen . The cells we e
disagg ega ed, ixed and pe meabilized as desc ibed abo e. The samples we e main ained a 4
◦
C in
washing bu e un il all cells we e collec ed, in o de o incuba e all he samples wi h he an ibodies
a once. Fluo escence alues we e analysed and eco ded and hen he posi i e cells we e collec ed,
and he RNA isola ed as desc ibed p e iously. RNA copies (co esponding o he VP2 sequence) pe
IPNV in ec ed cell we e quan i ied and co ela ed wi h he VP2 luo escence alues in he MFI uni s.
Fo his pu pose, he Pea son co ela ion es was pe o med using G aphPad P ism e sion 5.00
(G aphPad So wa e, Inc., La Jolla, CA, USA).
Appl. Sci. 2018,8, 1734 5 o 14
3. Resul s
3.1. Op imiza ion o he P ocedu e and Assessmen o he Reliabili y o he Da a
Fo he op imiza ion o he p ocedu e, and o e alua e i s abili y o disc imina e be ween di e en
i al concen a ions, ou mul iplici ies o in ec ion (MOI) we e es ed ( om 0.1 o 0.0001), and he
co ela ion be ween he i al doses and he di e en pa ame e s was assessed by eg ession analysis.
As shown in Table 1, he pe cen age o in ec ed cells dec eased wi h he MOI and bo h pa ame e s
co ela ed signi ican ly (R
2
= 0.9882; Figu e 1A). Using he ob ained MESF da a and he pe cen ages
o posi i e cells, he co esponding MFI alues we e calcula ed, which as expec ed also showed a
signi ican co ela ion wi h he in ec ed cells (R2= 0.9682; Figu e 1B).
Appl. Sci. 2018, 8, x FOR PEER REVIEW 5 o 16
analysis. As shown in Table 1, he pe cen age o in ec ed cells dec eased wi h he MOI and bo h
pa ame e s co ela ed signi ican ly (R2 = 0.9882; Figu e 1A). Using he ob ained MESF da a and he
pe cen ages o posi i e cells, he co esponding MFI alues we e calcula ed, which as expec ed also
showed a signi ican co ela ion wi h he in ec ed cells (R2 = 0.9682; Figu e 1B).
Figu e 1. Co ela ion be ween mul iplici y o in ec ion (MOI), in ec ed cells, Mean o Fluo escence
In ensi y (MFI) and RNA molecules. Each da a co esponds o a single da a poin . (A) Reg ession
be ween MOI and pe cen age o in ec ed cells; (B) Reg ession be ween MOI and MFI; (C) Reg ession
be ween MFI and i al RNA copies pe cell; (D) Reg ession be ween MFI and i al RNA copies pe
posi i e cells. In g aphs wi h 2 equa ions, he i s one co esponds o a eg ession line equa ion (and R2
alue) using he alues shown on bo h axes; he second one co esponds o a eg ession line equa ion (and
R2 alue) using loga i hm o he alues shown on bo h axes. Dashed lines: eg ession cu es.
In addi ion, he i al RNA was ex ac ed om a numbe o posi i e cells and quan i ied by RT-
qPCR o de e mine he numbe o RNA copies pe cell. The o al numbe o copies (pe 100 cells) was
calcula ed mul iplying ha alue by he pe cen age o in ec ed cells (Table 1). As shown in Figu e
1C,D, a clea co ela ion was obse ed be ween ei he pa ame e s (RNA copies pe cell and pe all
posi i e cells) and he MFI alues, ollowing a second-deg ee eg ession wi h R2 alues 0.9734 and
0.9998, espec i ely.
Table 1. P elimina y assays o e alua e he pe o mance o he me hod o disc imina ion be ween
di e en i us concen a ions.
Fluo escence Pa ame e s Vi al RNA
Sample %
+Cells 1 AFI
2 MESF
3 MFI
4 Pe Cell
5 Pe All
+Cells 6
FITC+
MOI 0.1 65.40 1025 373,696 24,439,718.4 402,807 26,343,577.8
MOI 0.01 8.81 34,962 919,698 8,102,539.4 38,615 340,198.2
MOI 0.001 1.82 4537 2
,
386
,
761 4
,
343
,
905.0 33
,
104 60
,
249.3
MOI 0.0001 0.12 211,266 6,912,796 829,535.5 21,675 2601.0
1 % +Cells: Pe cen age o posi i e cells; 2 AFI: a bi a y luo escence in ensi y uni s (geome ic mean alues);
3 MESF: Molecules o Equi alen Soluble Fluo och ome uni s, no malized wi h espec o he nega i e
con ol (C-MOCK, Mock in ec ed cells); 4 MFI: Mean Fluo escence In ensi y o he in ec ed cells, calcula ed
mul iplying he MESF da a by he co esponding pe cen ages o in ec ed cells; 5 Numbe o RNA copies
Figu e 1.
Co ela ion be ween mul iplici y o in ec ion (MOI), in ec ed cells, Mean o Fluo escence
In ensi y (MFI) and RNA molecules. Each da a co esponds o a single da a poin . (
A
) Reg ession
be ween MOI and pe cen age o in ec ed cells; (
B
) Reg ession be ween MOI and MFI; (
C
) Reg ession
be ween MFI and i al RNA copies pe cell; (
D
) Reg ession be ween MFI and i al RNA copies pe
posi i e cells. In g aphs wi h 2 equa ions, he i s one co esponds o a eg ession line equa ion (and R
2
alue) using he alues shown on bo h axes; he second one co esponds o a eg ession line equa ion
(and R2 alue) using loga i hm o he alues shown on bo h axes. Dashed lines: eg ession cu es.
Table 1.
P elimina y assays o e alua e he pe o mance o he me hod o disc imina ion be ween
di e en i us concen a ions.
Fluo escence Pa ame e s Vi al RNA
Sample %+Cells 1AFI 2MESF 3MFI 4Pe Cell 5Pe All +Cells 6
FITC+
MOI 0.1 65.40 1025 373,696 24,439,718.4 402,807 26,343,577.8
MOI 0.01 8.81 34,962 919,698 8,102,539.4 38,615 340,198.2
MOI 0.001 1.82 4537 2,386,761 4,343,905.0 33,104 60,249.3
MOI 0.0001 0.12 211,266 6,912,796 829,535.5 21,675 2601.0
1
%
+
Cells: Pe cen age o posi i e cells;
2
AFI: a bi a y luo escence in ensi y uni s (geome ic mean alues);
3
MESF:
Molecules o Equi alen Soluble Fluo och ome uni s, no malized wi h espec o he nega i e con ol (C-MOCK,
Mock in ec ed cells);
4
MFI: Mean Fluo escence In ensi y o he in ec ed cells, calcula ed mul iplying he MESF
da a by he co esponding pe cen ages o in ec ed cells;
5
Numbe o RNA copies pe cell de e mined by RT-qPCR
using
in i o
ansc ibed i al RNA as s anda d;
6
Numbe o RNA copies pe all posi i e in ec ed cells. Each da a
co esponds o a single da a poin .
Appl. Sci. 2018,8, 1734 6 o 14
In addi ion, he i al RNA was ex ac ed om a numbe o posi i e cells and quan i ied by
RT-qPCR o de e mine he numbe o RNA copies pe cell. The o al numbe o copies (pe 100 cells)
was calcula ed mul iplying ha alue by he pe cen age o in ec ed cells (Table 1). As shown in
Figu e 1C,D, a clea co ela ion was obse ed be ween ei he pa ame e s (RNA copies pe cell and pe
all posi i e cells) and he MFI alues, ollowing a second-deg ee eg ession wi h R
2
alues 0.9734 and
0.9998, espec i ely.
3.2. Repea abili y and Rep oducibili y
To assess eliabili y in e ms o epea abili y and ep oducibili y, he p ocedu e was es ed wi h
BF-2 in ec ed cells (24 h p.i.), using h ee eplicas o e alua e epea abili y, and he assay was epea ed
in h ee di e en days o es ep oducibili y.
Since he MESF alues o he no malized beads a e p o ided by he manu ac u e as s anda d
uni s, hei epea abili y and ep oducibili y we e e alua ed using he obse ed AFI alues. As shown
in Table 2, he CV alues co esponding o bo h epea abili y and ep oducibili y we e always below
10% (on all 3 days). In addi ion, he eliabili y o he s anda d cu es was demons a ed by he high
co ela ion alues (R
2
> 0.99) o he eg ession lines be ween he expec ed MESF (as indica ed by
he manu ac u e ) and he obse ed AFI alues (Figu e 2). In addi ion, he epea abili y o he h ee
luo escence pa ame e s (AFI, MESF, and MFI) o he IPNV in ec ed cells was high, wi h CV alues
≤
7.2% in all cases. The same esul was obse ed in he ep oducibili y o he AFI and MESF da a;
al hough, in he case o MFI he CV co esponding o ep oducibili y was sligh ly highe (CV = 11.5%;
Table 2).
Appl. Sci. 2018, 8, x FOR PEER REVIEW 6 o 16
pe cell de e mined by RT-qPCR using in i o ansc ibed i al RNA as s anda d; 6 Numbe o RNA
copies pe all posi i e in ec ed cells. Each da a co esponds o a single da a poin .
3.2. Repea abili y and Rep oducibili y
To assess eliabili y in e ms o epea abili y and ep oducibili y, he p ocedu e was es ed wi h
BF-2 in ec ed cells (24 h p.i.), using h ee eplicas o e alua e epea abili y, and he assay was epea ed
in h ee di e en days o es ep oducibili y.
Since he MESF alues o he no malized beads a e p o ided by he manu ac u e as s anda d
uni s, hei epea abili y and ep oducibili y we e e alua ed using he obse ed AFI alues. As
shown in Table 2, he CV alues co esponding o bo h epea abili y and ep oducibili y we e always
below 10% (on all 3 days). In addi ion, he eliabili y o he s anda d cu es was demons a ed by he
high co ela ion alues (R2 > 0.99) o he eg ession lines be ween he expec ed MESF (as indica ed by
he manu ac u e ) and he obse ed AFI alues (Figu e 2). In addi ion, he epea abili y o he h ee
luo escence pa ame e s (AFI, MESF, and MFI) o he IPNV in ec ed cells was high, wi h CV alues
≤ 7.2% in all cases. The same esul was obse ed in he ep oducibili y o he AFI and MESF da a;
al hough, in he case o MFI he CV co esponding o ep oducibili y was sligh ly highe (CV = 11.5%;
Table 2).
Figu e 2. Repea abili y and ep oducibili y. Linea eg ession be ween he obse ed luo escence
(AFI) and he expec ed MESF epo ed by he manu ac u e o he beads employed as s anda d.
3.3. Reliabili y o he qFCM o Vi al Ti a ion
To e alua e he p ocedu e o i al quan i ica ion, qFCM was applied using en- old dilu ions
o he i us ( i a ed by wo me hods: plaque o ming uni s and endpoin dilu ion), and he esul s
a e shown in Table 3. Su p isingly, he MESF alues co esponding o he lowes MOIs (0.00001 and
0.000001) we e highe han he p e ious ones; howe e , his was co ec ed when he pe cen age o
in ec ed cells a each MOI was conside ed o calcula e he MFI alues.
Figu e 2.
Repea abili y and ep oducibili y. Linea eg ession be ween he obse ed luo escence (AFI)
and he expec ed MESF epo ed by he manu ac u e o he beads employed as s anda d.
3.3. Reliabili y o he qFCM o Vi al Ti a ion
To e alua e he p ocedu e o i al quan i ica ion, qFCM was applied using en- old dilu ions
o he i us ( i a ed by wo me hods: plaque o ming uni s and endpoin dilu ion), and he esul s
a e shown in Table 3. Su p isingly, he MESF alues co esponding o he lowes MOIs (0.00001 and
0.000001) we e highe han he p e ious ones; howe e , his was co ec ed when he pe cen age o
in ec ed cells a each MOI was conside ed o calcula e he MFI alues.
Appl. Sci. 2018,8, 1734 7 o 14
Table 2. Repea abili y and Rep oducibili y.
Repea abili y Day 1
AFI MESF
Replica 1 Replica 2 Replica 3 Mean SD CV Replica 1 Replica 2 Replica 3 Mean SD CV
Beads
B1 15.07 15.14 15.39 15.20 0.17 1.1 1929 1929 0 0
B2 36.10 36.30 37.39 36.60 0.70 1.9 8750 8750 0 0
B3 127.85 127.91 134.23 130.00 3.67 2.8 34,864 34,864 0 0
B4 406.90 425.17 463.92 432.00 29.12 6.7 127,311 127,311 0 0
B5 1197.54 1219.18 1325.49
1247.40
68.49 5.5 421,992 421,992 0 0
Samples
Blank 102.50 105.80 112.70 107.00 5.20 4.9 23,579.62 24,483.96 26,391.86 24,818.48 1435.66 5.8
C-MOCK 140.20 131.50 145.40 139.03 7.02 5.1 34,205.24 31,699.19 35,717.23 33,873.88 2029.41 6.0
FITC +1647.40 1656.80 1795.20
1699.80
82.75 4.9 638,252.09 642,579.82 706,821.61 662,551.17 38,400.90 5.8
Repea abili y Day 2
AFI MESF
Replica 1 Replica 2 Replica 3 Mean SD CV Replica 1 Replica 2 Replica 3 Mean SD CV
Beads
B1 18.41 17.05 17.64 17.70 0.68 3.9 1929 1929 0 0
B2 38.67 36.72 38.01 37.80 0.99 2.6 8750 8750 0 0
B3 152.00 129.09 117.91 335.00 7.91 5.9 34,864 34,864 0 0
B4 457.93 487.65 449.49 465.02 20.04 4.3 127,311 127,311 0 0
B5 1495.67 1410.55 1263.89
1390.04
117.24 8.4 421,992 421,992 0 0
Samples
Blank 96.51 95.59 90.93 94.30 2.99 3.2 20,011.7 19,785.44 18,645.63 19,480.93 732.18 3.8
C-MOCK 178.71 145.90 130.96 151.90 24.43 16.1 41,586.38 32,686.35 28,751.93 34,341.55 6575.37 19.1
FITC +1847.76 1949.35 1761.12
1852.70
133.10 7.2 665,827.32 709,507.84 628,507.58 668,087.58 40,337.94 6.0
Repea abili y Day 3
AFI MESF
Replica 1 Replica 2 Replica 3 Mean SD CV Replica 1 Replica 2 Replica 3 Mean SD CV
Beads
B1 16.69 16.28 15.34 16.10 0.69 4.3 1929 1929 0 0
B2 41.19 38.93 37.48 39.20 1.87 4.8 8750 8750 0 0
B3 145.51 130.46 128.15 135.04 10.00 7.4 34,864 34,864 0 0
B4 490.08 543.75 434.09 489.31 44.84 9.2 127,311 127,311 0 0
B5 1560.61 1544.34 1314.78
1473.24
137.47 9.3 421,992 421,992 0 0
Appl. Sci. 2018,8, 1734 8 o 14
Table 2. Con .
Samples
Blank 135.17 135.82 128.07 133.00 4.3 3.2 29,451.79 29,615.92 27,667.32 28,911.68 1080.77 3.7
C-MOCK 184.52 186.45 169.11 180.01 9.50 5.3 42,236.19 42,748.36 38,177.84 46.032.39 2504.07 6.1
FITC +1980.12 1906.03 1818.85
1901.70
80.72 4.2 660,066.13 631,542.06 598,203.17 629,937.12 30,962.69 4.9
Rep oducibili y
Day 1 AFI MESF
Beads AFI MESF AFI MESF AFI MESF Mean SD CV Mean SD CV
B1 15.2 1929.0 17.7 1929.0 16.1 1929.0 16.33 1.3 7.8 1929.00 0.00 0.00
B2 36.6 8750.0 37.8 8750.0 39.2 8750.0 37.87 1.3 3.4 8750.00 0.00 0.00
B3 130.0 34,864.0 133.0 34,864.0 135.0 34,864.0 132.67 2.5 1.9 34,864.00 0.00 0.00
B4 432.0 127,311.0 465.0 127,311.0 489.0 127,311.0 462.00 28.8 6.2 127,311.00 0.00 0.00
B5 1247.4 421,992.0 1390.0 421,992.0 1473.0 421,992.0 1370.23 114.2 8.3 421,992.00 0.00 0.00
Blank 107.00 24,818.48 94.34 19,480.93 133.02 28,911.68 111.45 19.7 17.7 24,403.69 4729.0 19.4
C-MOCK 139.03 33,818.88 151.86 34,341.55 180.03 41,054.13 156.97 21.0 13.4 34,215.67 4017.3 11.7
FITC+1699.80 662,551.17 1852.74 668,087.58 1901.67 629,937.12 1818.07 105.3 5.8 635,525.29 20,614.7 3.2
(MFI) (24,050,607.6) (25,387,328.0) (29,859,019.5)
(26,432,318.4)
(3,041,943) (11.5)
AFI, a bi a y luo escence in ensi y uni s (geome ic mean alues); MESF, Molecules o Equi alen Soluble Fluo och ome uni s; SD, S anda d de ia ion; CV, Coe icien o a ia ion
(SD/a e age ×100); C-MOCK, Nega i e con ol (Mock in ec ed cells); MFI, Mean Fluo escence In ensi y o he in ec ed cells.
Table 3. Reliabili y o he qFCM o i al i a ion.
Inocula ed Vi us 1Assay 1 Assay 2
MOI TCID50/mL p u/mL %+Cells 2AFI 3MESF 4MFI 5%+Cells 2AFI MESF MFI
0.1 1×1062.6 ×10666.4 7901 206,112 13,685,836.8 82.6 5761 241,276
19,929,423.9
0.01 1×1052.6 ×10554.1 6641 173,314 9,376,287.4 67.8 4154 166,502
11,288,862.9
0.001 1×1042.6 ×10413.3 6967 181,801 2,417,953.3 23.7 1655 58,628.7 1,389,500.3
0.0001 1×1032.6 ×10317.6 6344 165,581 2,914,225.6 18.6 1395 48,297.5 898,333.3
0.00001 1×1022.6 ×1021.87 13,615 324,712 607,211.4 2.63 874 28,418.9 74,741.8
0.000001 1×1012.6 ×1011.97 11,188 291,618 574,487.5 2.76 963 31,723 87,555.6
1
Ti a ed by endpoin dilu ion (TCID
50
) and plaque o ming uni s (p u);
2
%
+
Cells, Pe cen age o posi i e cells;
3
AFI, a bi a y luo escence in ensi y uni s (geome ic mean alues);
4
MESF, Molecules o Equi alen Soluble Fluo och ome uni s, no malized wi h espec o he nega i e con ol (C-MOCK, Mock in ec ed cells);
5
MFI, Mean Fluo escence In ensi y o he
in ec ed cells, calcula ed mul iplying he MESF da a by he co esponding pe cen ages o in ec ed cells. Each da a co esponds o a single da a poin .
Appl. Sci. 2018,8, 1734 9 o 14
On he o he hand, a signi ican co ela ion (R
2
= 0.9559, assay 1; R
2
= 0.9577, assay 2) was
obse ed be ween he i al i e and he pe cen age o in ec ed cells, by a second-deg ee polynomial
eg ession (y = 2.8979x
2−
6.7267x + 5466, assay 1; y = 3.3423x
2−
6.2588x + 4.229, assay 2) (da a no
shown). Mo e impo an ly, a clea co ela ion be ween i al i es and he MFI alues was obse ed,
also by a second-deg ee polynomial eg ession (Figu e 3).
Appl. Sci. 2018, 8, x FOR PEER REVIEW 10 o 16
On he o he hand, a signi ican co ela ion (R2 = 0.9559, assay 1; R2 = 0.9577, assay 2) was
obse ed be ween he i al i e and he pe cen age o in ec ed cells, by a second-deg ee polynomial
eg ession (y = 2.8979x2 − 6.7267x + 5466, assay 1; y = 3.3423x2 − 6.2588x + 4.229, assay 2) (da a no
shown). Mo e impo an ly, a clea co ela ion be ween i al i es and he MFI alues was obse ed,
also by a second-deg ee polynomial eg ession (Figu e 3).
Figu e 3. Reliabili y o qFCM o i al i a ion. Second deg ee polynomial eg ession be ween he
i al i e (in p u and TCID50) and luo escence (in MFI). Each da a co esponds o a single da a poin .
Finally, o compa e he limi o de ec ion o qFCM wi h ha o he adi ional me hod in ol ing
i us isola ion, he same i al dilu ions employed in qFCM we e subjec ed o e-isola ion in BF-2,
obse ing ha e-isola ion was no possible om he las dilu ion (co esponding o 101 TCID50/mL
o 2.6 × 101 p u/mL).
Figu e 3.
Reliabili y o qFCM o i al i a ion. Second deg ee polynomial eg ession be ween he
i al i e (in p u and TCID
50
) and luo escence (in MFI). Each da a co esponds o a single da a poin .
Finally, o compa e he limi o de ec ion o qFCM wi h ha o he adi ional me hod in ol ing
i us isola ion, he same i al dilu ions employed in qFCM we e subjec ed o e-isola ion in BF-2,
obse ing ha e-isola ion was no possible om he las dilu ion (co esponding o 10
1
TCID
50
/mL
o 2.6 ×101p u/mL).
