development of strand-specific real-time rt-pcr to

Research ArticleDevelopment of Strand-Specific Real-Time RT-PCRto Distinguish Viral RNAs during Newcastle DiseaseVirus Infection

Xusheng Qiu Yang Yu Shengqing Yu Yuan Zhan Nana Wei Cuiping SongYingjie Sun Lei Tan and Chan Ding

Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences (CAAS) 518 Ziyue Road Shanghai 200241 China

Correspondence should be addressed to Chan Ding shoveldeenshvriaccn

Received 5 June 2014 Revised 12 August 2014 Accepted 12 August 2014 Published 14 October 2014

Academic Editor Peirong Jiao

Copyright copy 2014 Xusheng Qiu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Newcastle disease virus (NDV) causes large losses in the global fowl industry To better understand NDV replication andtranscription cycle quantitative detectionmethods for distinguishingNDV genomic RNA (gRNA) antigenomic RNA (cRNA) andmessenger RNA (mRNA) in NDV-infected cells are indispensibleThree reverse transcription primers were designed to specificallytarget the nucleoprotein (NP) region of gRNA cRNA and NPmRNA and a corresponding real-time RT-PCR assay was developedto simultaneously quantify the three types of RNAs in NDV-infected cells This method showed very good specificity sensitivityand reproducibility The detection range of the assay was between 55 times 102 and 11 times 109 copies120583L of the target gene Thesemethodswere applied to investigate the dynamics of the gRNA cRNA andmRNA synthesis inNDVLa Sota infectedDF-1 cellsTheresults showed that the copy numbers of viral gRNA cRNA and NPmRNA all exponentially increased in the beginningThe viralRNA copy number then plateaued at 10 h postinfection and gradually decreased from 16 h postinfection No synthesis priority wasobserved between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection Howeverthe cRNA accumulated more rapidly than gRNA as the cRNA copy number was three- to tenfold higher than gRNA starting from2 h postinfection Conclusion A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cellswas developed for the first time The development of this assay will be helpful for further studies on the pathogenesis and controlstrategies of NDV

1 Introduction

Newcastle disease virus (NDV) is an economically dev-astating pathogen in the global poultry industry [1 2]NDV a member of the family Paramyxoviridae (genusAvulavirus in subfamily Paramyxovirinae) [3] has a single-stranded nonsegmented negative-sense RNA genome ofapproximately 15 kb Six viral structural proteins are encodedfrom its genome a nucleoprotein (NP) phosphoprotein(P) matrix protein (M) fusion protein (F) haemagglutinin-neuraminidase (HN) and large protein (L) [4ndash7] In additionto those viral gene products two more nonstructural pro-teins V and W are encoded from the P gene via an RNA-editing mechanism [8ndash10]

All NDV viral mRNAs are transcribed from NDVgenomic RNA by using the viral RNA-dependent RNA

polymerase (vRdRp) assembled fromNP P andLproteins [1112]TheLprotein is the core element of vRdRp containing thepolymerase activity as well as capping and polyadenylationactivities [13ndash17]The P protein acts as a bridge between the Lprotein and NP protein which encapsidates the viral genome[18] The NP-P complex is thought to be the substrate usedby the RdRp to initiate the encapsidation of the nascent RNAchain during viral replication [19 20]

In the early phase of NDV infection the HN protein ofNDV attached to the surface of cells and the following fusionbetween viral and cell membranes was mediated by the Fprotein [21] Subsequently the NP-wrapped viral genomicRNA as well as other viral components was released intothe cytoplasm [22 23] The NP protein encapsidates viralgRNA and cRNA to resist host nucleases and also mediatesviral RNA replication and transcription by RdRp [12 24 25]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 934851 10 pageshttpdxdoiorg1011552014934851

2 The Scientific World Journal

The (minus) viral genomic RNA termed gRNA is a template forsynthesis of mRNA and the intermediate (+) viral genomicRNA (termed cRNA in this study) the latter was used as thetemplate for gRNA synthesis [7 26 27] The cRNAs serve asreplicative intermediates for gRNA duplication and are notknown to encode viral proteins [28]

The amounts of viral gRNA cRNA and mRNA report-edly change in a regular pattern in cells infected with themeasles virus influenza virus and Nipah virus [29ndash32]suggesting these RNA viruses regulate viral replication andtranscription through specific molecular mechanisms Inthis study a strand-specific real-time RT-PCR assay wasdeveloped to distinguish viral gRNA cRNA and mRNAlevels in NDV-infected cells This will aid studies into themolecular mechanisms of viral replication and transcriptionviral pathogenesis and control strategies for NDV

2 Materials and Methods

21 Cell and Virus DF-1 cells were obtained from the Amer-ican Type Culture Collection (ATCC Manassas VA USA)and grown in RPMI-1640 media (10 fetal bovine serum2mM L-glutamine 100 IUmL penicillin 100 120583gmL strepto-mycin and 10mM HEPES buffer) at 37∘C and 5 CO

2 Cell

culture reagents were obtained from Gibco (Rockville MDUSA)

NDVLa Sota strainwas obtained from theChina Instituteof Veterinary Drug Control (Beijing China) and propagatedin 9- to 11-day-old specific-pathogen-free (SPF) embryosTheinfected allantoic fluid was harvested between 84 and 120 hThe virus was determined for the 50 tissue culture infectivedose (TCID

50) and kept at minus80∘C before use

22 Virus Infection The incubation time for the virus infec-tion was optimized first DF1 cells (3 times 105 cells) werecultivated in 60mm dishes at 37∘C and 5 CO

2 For La

Sota infection a monolayer of DF1 cells was washed withphosphate-buffered saline (PBS) three times and 1mL ofserum-free 1640 medium containing NDV La Sota at amultiplicity of infection (MOI) of 001 was then added At5 s 1min 5min 10min 15min 20min 25min 30min and60min postinfection (pi) the cells were washed again withPBS to remove unbound virus particles and harvested forRNA extraction and subjected to real-time RT-PCR Theoptimal incubation time for NDV infection was based onwhen the cDNAs of gRNA cRNA and mRNA of NP startedto stably accumulate The quantitation cycle (119862

119902) values of

each RNA at this time point were considered residual viralRNAs after incubation and set as baseline values Accordingto the optimized incubation time determined DF1 cells wereinfectedwith La Sota at anMOI of 001 After incubation cellswere washed with PBS and then grown in 4mL 1640mediumcontaining 1 (vv) fetal calf serum at 37∘C and 5 CO

2 The

cells and supernatant were harvested at different time pointsfor the assay

23 RNA Extraction and Strand-Specific Reverse Transcrip-tion After infection with NDV La Sota the supernatant and

cells were collected and thoroughly mixed and 500120583L ofthe mixture was lysed with 1mL TRIZol reagent (Invitro-gen Carlsbad CA USA) The total RNA of the virus wasextracted according to the manufacturerrsquos instructions TheRNA pellets were resuspended in 50 120583L RNase-free waterafter washing with 70 ethanol To degrade cellular DNADNase I was added to the samples at a final concentrationof 1 U120583L at 37∘C for 30min and then inactivated at 65∘C for10min in the presence of 5mM EDTA

Strand-specific reverse transcription of NDV gRNAcRNA and mRNA was performed with specifically designedreverse transcription primer sets (Figure 1) To quantify thethree kinds of cDNAs with one pair of primers reverse tran-scription primers specific for gRNA cRNA or mRNA weredesigned using the Lasergene software suite (DNASTARMadison WI USA)

The sequence of the gRNA-specific reverse transcriptionprimer PLa-Gwas 51015840-ACGATAAAAGGCGAAGGAGCA-31015840 (genomic position 22ndash44 nt) which initiated synthesis ofthe unique leader sequence of the NDV genome

The sequence of primer PLa-R specific to the P gene ofviral cRNA was 51015840-GTC GTT TGC TCG GGT GTG GAT G-31015840 (genomic position 2 119-2 139 nt)

The extracted mRNA segments involving viral mRNAwere reverse-transcribed byPLa-18V (51015840-TTTTTTTTTTTTTTT TTT AGC-31015840)

Each 10 120583L of the above RNA samples was reverse-transcribed by respective primers in the presence of M-MLV reverse transcriptase (Promega Madison WI USA)according to the protocol Briefly 200 U M-MLV reversetranscriptase 5120583L 5 times RT buffer 10 120583L RNA sample 10mmolspecific RT primer 10mmol dNTPs and ultrapure water(Gibco BRL Gaithersburg MD USA) were added to a finalvolume of 25 120583L and mixed The mixture was incubated at42∘C for 60min and then 75∘C for 15min to denature reversetranscriptase The reverse transcripts were precipitated usingtwo volumes of ethanol Finally the yielded pellet wasdissolved in 20120583L of ultrapure water (Gibco BRL) and keptat minus70∘C until use

24 Real-Time Quantitative Polymerase ChainReaction (PCR) Assay

241 Construction of Plasmid T-NP as a Standard for theAssay To construct a standard curve for the linear corre-lation between 119862

119902values and molecular numbers of tar-

get cDNAs plasmid T-NP was constructed and used as astandard Genomic RNA was reverse-transcribed by randomhexamer primers and 5 120583L of the resultant cDNA fragmentswas then added into a 50120583L PCR reaction system containing25U TaKaRa LA Taq 5 120583L 10 times LA PCR Buffer II (Mg2+Plus) 1120583L 20120583M former primer PNPF 1 120583L 20120583M reversetranscription- (RT-) primer PNPR and 8 120583L 25mM dNTPmixtureThe primer pairs of PNPF (51015840-CAG TGA TGACCCAGA AGA TAG ATG-31015840) and PNPR (51015840-CGC AAA GCTCAT CTG GTC ACT ATC-31015840) were used to amplify the NPgene The PCR cycle conditions were 95∘C 1min 25 cycles of94∘C 30 s 55∘C 30 s and 72∘C 90 s followed by 72∘C 10min

The Scientific World Journal 3

+++++++++++++++

+++++++++++++

++++++++++

+++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++

Le NP PVW M F HN L Tr

Cap

Cap

Cap

Cap

Cap

Cap

56ndash1792 1804ndash3244 3256ndash4487 4498ndash6279 6412ndash8145 8370ndash15073

3998400

5998400 3998400

5998400Genomic RNA gRNA

Antigenomic RNA cRNA

M

mRNA

P mRNA

F

mRNA

HN

mRNA

LmRNA

NP

mRNAminusAn

minusAn

minusAn

minusAn

minusAn

minusAn

(a)

463 607

Le NP

Le NP

NP

P

P

C

gRNA

mRNA

cRNA

Pla-18T

Pla-G

Pla-R

Pla-rt13

Pla-rt14

Pla-rt13

Pla-rt14

Pla-rt14

Pla-rt13

Poly(A)n

3998400

5998400

39984005998400

3998400

5998400

(b)

Figure 1 Schematic chart of primer design (a) Schematic view of NDV genomic organization main RNA species and nucleotide positions(b) Binding position of RT-qPCR primers Red arrows represent the locations of primers for reverse transcription Black arrows represent thelocations of primers for real-time RT-PCR amplification

The PCR product was subcloned into the pGEM-Teasy vector (Promega) to construct the plasmid T-NP andidentified by nucleotide sequence analysis Plasmid DNAwas purified using QIAGEN Plasmid Midi Kits and thenthe concentration and purity were determined by the ND-1000 spectrophotometer (NanoDrop Technologies Inc)Thepurified plasmid T-NP with an A260280 ratio of 180qualified as a standard

242 Optimization of the Real-Time Quantitative PCR Pro-cedure To obtain suitable amplification efficiency threepairs of primers including Pla-rt11Pla-rt12 Pla-rt13Pla-rt14 and Pla-rt21Pla-rt22 pairs (Table 1) were designedaccording to ldquoMIQE guidelines for PCRrdquo [33 34] based onthe cDNA sequence of the NDV La Sota strain (GenBankID LaSota AF077761) and used for NP gene amplificationwith viral cDNA and T-NP as templates Following the

manufacturerrsquos instructions quantitative PCR (qPCR) wasperformed using Maxima SYBR Green dye (Fermentas GlenBurnie MD USA) in a PCR thermocycler from EppendorfAG (Hamburg Germany) operated by the Mastercyclerep realplex system The qPCR reaction mixture comprised125 120583L of 2x Maxima SYBR Green qPCR Master Mix 10 120583Lof reverse transcription product and 25 pmol of each real-time primer made up to a final volume of 25 120583L withultrapure water (Gibco BRL) The cycle conditions of qPCRwere 95∘C 5min followed by 40 cycles of 95∘C 15 s 60∘C30 s and 72∘C 30 s Nuclease-free ultrapure water (GibcoBRL) was used as a template for the negative control Thespecificity of the qPCR primers was monitored with meltingcurve analysis

243 Linear Regression Analysis To establish the statisticalrelationship between119862

119902values and themolecular numbers of


Table 1 Primers used in this study

Primers Sequences (51015840-31015840) Location3 Fragment designationPla1F1 TATCCAGGCTCAAGTATGGGTCACA 622ndash646 Pla1Pla1R2 CCTTGGTCTTGCCTTGTGGGATTG 1999ndash2022Pla2F ATATTCAGAGATCAGGGCAAGTC 1819ndash1841 Pla2Pla2R GTTTGCCACAACCCTACAGC 3708ndash3724Pla3F AAAGCTGTAGGGTTGTGG 3705ndash3727 Pla3Pla3R TTGGCGATGACTGAACCT 5704ndash5721Pla4F AGGCGCACTTACTACACCATA 5671ndash5691 Pla4Pla4R TGCTGTATGCGTTTCCCACCA 7502ndash7525Pla5F TACAGCAGGCTATCTTATCT 7517ndash7536 Pla5Pla5R TGAGTCACGGATTCTGCT 9370ndash9387Pla6F GTGACTCATGCAATCGCTACT 9377ndash9400 Pla6Pla6R CTAATTGGGCAGGAGTCAGA 11106ndash11126Pla7F TGCAGAGATCAAGCGACTA 11197ndash11215 Pla7Pla7R AGCATCTTCTCATTCAGGTTATC 13105ndash13128Pla8F TATCCCGGTTATGCTGTC 13137ndash13154 Pla8Pla8R TAAGACATTTATTTGAGTTCG 15103ndash15122Pla-rt11 CAATAGGAGTGGAGTGTCTGA 466ndash486 Real timePla-rt12 TCCTCTCCAGGGTATCGGTGA 549ndash614Pla-rt13 CAACAATAGGAGTGGAGTGTCTGA 463ndash486 Real timePla-rt14 CAGGGTATCGGTGATGTCTTCT 586ndash607Pla-rt21 TCTGTACTGTACTTGACTCGTGCTC 14903ndash14927 Real timePla-rt22 CTGTAATATCCGTTGACTGCATTGCCT 14953ndash149791F represents forward primer2R represents reverse transcription- (RT-) primer3The location was based on the genomic sequence of NDV La Sota (GenBank ID LaSota AF077761)

samples 5 ng120583L T-NP plasmid (A260280 ratio = 180) wasfivefold serially diluted and quantitated with the primer pairsof Pla-rt13 and Pla-rt14 A standard curve was then generatedby plotting 119862

119902values against logarithmic molecular numbers

of the standard and linear regression analysis was conductedto obtain a linear regression equation

244 Specificity of the Assay To determine the specificity ofthe Pla-rt13 and Pla-rt14 primers RT-PCR was performedwith TaKaRa LA Taq to obtain DNA fragments comprisingportions of the viral genome other than the NP gene Eightprimer pairs (Table 1) targeting genes other than NP weredesigned using the Lasergene software suite (DNASTAR) andused for PCR amplification of NDV genes other than NP

The PCR products were purified and their concentrationand purity were tested using a ND-1000 spectrophotometer(NanoDrop Technologies Inc) Only the PCR products withan A260280 ratio of 180 qualified for the study The eightPCR products were tenfold serially diluted and quantifiedThe 119862

119902values of PCR products with different concentrations

were compared with that of T-NP and the negative control(nuclease-free ultrapure water)

245 Sensitivity and Reproducibility of the Assay To con-firm assay sensitivity and reproducibility the T-NP plasmid(5 ng120583L) was tenfold serially diluted to 00005 ng120583L andused for the detection The amplification efficiency and

coefficient of variation (CV) of the qPCR reaction werecompared between different template concentrations andthe detection range was determined The experiment wasperformed in triplicate

246 Quantitative Analysis of Viral RNA Kinetics Total viralRNAs extracted fromNDV-infected cells at 1 2 3 4 5 6 8 1012 16 20 24 36 and 48 hpi were quantitatively analyzedThesamples were first reverse-transcribed with primers PLa-GPLa-R or PT-18V The resultant cDNA products (1 120583L cDNAproductwas used for each reaction)were then quantifiedwiththe primer pairs of Pla-rt13 and Pla-rt14 The total RNA ofthe DF-1 cells was used as a negative control Consideringthe interference of other ingredients in each sample theabsorption of cellular RNA harvested just after 20min wasalso detected and set as base line The values obtained by thereal-time RT-PCR assay were analyzed by linear regressionand the kinetic curves of gRNA cRNA and mRNA in LaSota-infected DF1 cells were generated

3 Results

31 Establishment and Validation of a Real-Time RT-qPCRAssay for NDV Three pairs of primers including Pla-rt11Pla-rt12 Pla-rt13Pla-rt14 and Pla-rt21Pla-rt22 pairs(Table 1) were initially designed and used for the amplifi-cation Neither nonspecific annealing nor primer dimer was


observed in the melting curves of these three primer pairsPla-rt13Pla-rt14 displayed an extremely stable amplificationefficiency around 1000 while those of Pla-rt1112 and Pla-rt 2122 were 0981 and 0987 respectively In this study thePla-rt13Pla-rt14 primer pairs were used for accurate qPCRamplification (Figure 1(a))

Eight PCR fragments Pla1ndashPla8 which comprised mostof the NDV genomic sequence were obtained at the expectedsizes After sequencing these fragments were confirmed andthen primed with Pla-rt13 and Pla-rt14 in a real-time PCRreactionNo significant cross-activitywas detectedwith thoseDNA fragments No cross-activity was shown with RNAextracts from uninfected cells or normal SPF chicken embryoallantoic fluidThus the developed qPCR assay demonstratedexcellent specificity

For quantitating cDNA molecules a standard curve wasgenerated with a fivefold serial dilution of the T-NP plasmidThe amplification efficiency was 101 and the concentrationat which linearity was retained in the standard curve was inthe range of 55 times 102ndash11 times 109 copies120583L (the detection rangeof the assay) Using linear regression a partial regressionline was calculated and the linear regression equation was119884 = minus3290 1119883 + 38922 (Figure 1(b)) An 1198772 value of 0998indicated strong linear correlations The highest 119862

119902value

of tested NTC (in this case ultrapure water was used fortemplate dilution) was 318 therefore test results with a 119862

119902

value lower than 2680 were considered positiveDF1 cells were infected with La Sota at a MOI of 001 and

the cells and supernatant were harvested at 48 hpi The totalRNAs were extracted and reverse-transcribed with primersPla-G Pla-R and Pla-18V respectively We designed RT-primer Pla-G to bind to the leader sequence of gRNA whichis absent in cRNA and mRNA [24 35] RT-primer Pla-R wasdesigned to bind to the P gene in front of the NP gene inviral antigenomic RNA therefore it is specific to viral cRNAThe PLa-18V primer is specific to all mRNAwith a polyA tailand viral gRNA and cRNA should not be reverse-transcribedAll the cDNAs of gRNA cRNA and mRNA were quantifiedwith same qPCR primer pairs specific for the NP gene Pla-rt13 and Pla-rt14 and similar amplification efficiency wasobserved in each reactionThe resultant cDNAs were tenfoldserially diluted and detected using RT-qPCR The 119862

119902values

of viral gRNA cRNA and NP gene mRNA ranged between874ndash3150 876ndash3146 and 926ndash3212 respectivelyThe linearregression analysis revealed 1198772 values in the range of 0991ndash0999 (Figure 2) The experiment was repeated three timesand each sample was performed in triplicate The coefficientof variation (CV) of 119862

119902values ranged from 20 to 50

between each experiment and less than 05 between eachrepeat of sample

32 Selection of the Incubation Time for La Sota InfectionTo investigate the interference from viral attachment anddetachment samples at different incubation times rang-ing from 5 s to 60min were detected and the molecularnumbers of intracellular RNA were quantified to determineRNA levels in the early phase of infection The attachmentand detachment during La Sota infection balanced 20min

after absorption (Figure 3) The viral RNA levels detectedindicated the quantity of viral particles that entered cellsby the adsorption time at the early phase of infection thelonger the adsorption time is the higher the level of gRNAdetected is However viral gRNA levels were low but stillslightly higher than that at instantaneous adsorption (5 s)when the adsorption timewas 20minThe results suggest thatinvasion and detachment may be balanced at this time Inaddition there was no significant difference in viral gRNAlevels between the adsorption times of 25 30 and 60minand gRNA levels at these time points were higher than thatat other time points The viral cRNA became detectable atthe adsorption time of 25min and then increased rapidlywhereas the NP genemRNAwas detectable at the adsorptiontime of 30min Therefore the incubation time for La Sotainfection was determined as 20min

33 Intracellular Kinetics of Viral RNAs during NDV InfectionThe gRNA cRNA and viral mRNA levels had similarincreasing trends in La Sota infected DF1 cells (Figure 4) Inthe earliest phase of NDV infection viral RNA increased ina geometric progression over the first 1 hpi the gRNA copynumber increased by more than one order of magnitudefrom 12 times 106 (the reference value at the adsorption time of20min) to 18times 107 whereas the cRNAandmRNAcopynum-bers increased from undetectable to almost the same level ofgRNA (18times 107 for cRNAand 28times 107 formRNA) Between 1and 8 hpi the viral RNA accumulated at an exponential rateand gRNA cRNA and mRNA copy numbers increased bythree orders of magnitude The synthesis of mRNA peakedat 10 hpi and decreased sharply from 20 hpi The synthesisof gRNA peaked at 12 hpi and declined from 16 hpi Thesynthesis of cRNA peaked at 20 hpi followed by its decreaseThroughout the course of infection cRNA levels began toexceed gRNA levels since 1 hpi and subsequently remained ata level 1ndash5 times as high as that of the gRNA levels (Figure 4)

4 Discussion

NDV has a (minus) single-stranded genomic RNA (gRNA)whereas the (+) single-stranded antigenomic RNA (cRNA)and mRNA are subsequently synthesized in cells upon NDVinfection (Figure 1(a)) The genomic RNA of negative RNAviruses is a template for the synthesis of cRNA and mRNA[28 36] The role of nascent cRNA is to serve as a templatefor the synthesis of gRNA Since no viral mRNA or protein isproduced from cRNA cRNA is considered an intermediatein the course of viral replication A switching mechanismhas been proposed in which viruses regulated the synthesisof different viral RNAs to facilitate infection NP M and Pproteins from influenzaA virus Sendai virus and themeaslesvirus have been reported to play an important role in theswitching mechanism between transcription and replication[27 29 37ndash40] However a similar regulation of viral RNAsynthesis was not explored in NDV until now

To investigate the kinetics of viral transcription and repli-cation in NDV-infected cells a reliable real-time RT-PCRassay for the differential quantification of viral gRNA cRNA


9492908886848280787674727068666462

Temperature (∘C)Threshold 33

minusdIdT

()

minus5

minus15

95

85

75

65

55

45

35

25

15

5

(a)

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10

y = minus32901x + 38922

R2 = 09982

Thre

shol

d cy

cle (C

T)

Log DNA copies per reaction

The standard curve graph of the PCI-NP real-time PCR assay

(b)

Figure 2The development of a real-time quantitative PCR assay for absolute quantification (a)Melting curve (b) linear regression equationThe amount of plasmid T-NP was converted into copy number via the following equation copy number = (amount of T-NP times 6022 times1023)(length of T-NP times 109times 660) The linearity of the standard curve was retained when the plasmid concentration was in the range of 55times 102ndash11 times 109 copies120583L The obtained linear regression equation was 119884 = minus32901119883 + 38922 (1198772 = 0998) The amplification efficiency was101

00

05

10

15

20

25

gRNAcRNA

mRNA

Different incubation times

gRNA cRNA and mRNA levels at different incubation times

log10

copi

esc

ell

5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m

Figure 3 Selection of the optimal adsorption time for La Sota infection gRNA cRNA and mRNA levels were measured at 5 s 1min 5min10min 15min 20min 25min and 30min after DF-1 cells were infected with the La Sota strain at an MOI of 1 The optimal adsorption timewas selected as 20min since the attachment and detachment of La Sota were balanced at this time point

and mRNA was developed in this study To simultaneouslydistinguish three kinds of viral RNA three primers PLa-GPLa-R and PLa-18V specific for the reverse transcription ofgRNA cRNA and mRNA respectively were designed andused for the assay Due to different transcription efficiencybetween specific and random RT-primers three kineticscurves for gRNA cRNA and mRNA were independentlyestablished to determine the synthesis pattern of these viralRNAs

The relative molecular numbers of gRNA and cRNA inthis studywere comparable for at least three potential reasons

First the sequences of the RT-primers Pla-G and Pla-R wereexactly complementary to the target gene Second Pla-G andPla-R were both located very close to the priming site of Pla-rt13 and Pla-rt-14 in the viral genomeThird Pla-G and Pla-Rdisplayed similar GC content

In following specificity test eight PCR fragments Pla 1 toPla 8 which covered most of the NDV genome except for theNP gene were used for testing the specificity of primers Theresults showed no cross-reaction with gene sequences otherthan NP since their 119862

119902values revealed levels far lower than

that of the interest gene (T-NP) even lower than the testing


gRNAcRNA

Hours postinfection

Kinetics of gRNA and cRNA in the La Sota infected DF1 cells

9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA

Kinetics of mRNA in the La Sota infected DF1 cells

9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)

Figure 4 Kinetics of gRNA cRNA and mRNA in La Sota infected DF-1 cells DF1 cells were incubated with NDV La Sota at an MOI of 1 for20min The gRNA cRNA and mRNA were quantitatively analyzed at 1 2 3 4 5 6 8 10 12 16 20 24 36 and 48 hpi (a) Kinetics of gRNAand cRNA (b) Kinetics of NP gene mRNA

limit Therefore by using a strand-specific real-time RT-PCRdeveloped in this study viral RNA accumulation kinetics inNDV-infected DF-1 cells were determined

In our study viral RNA synthesis was observed for thefirst time during the incubation of infection The copy num-ber of gRNA increased during incubation time indicatingan increase in the number of viruses entering cells withincreasing incubation time NDV cRNA was detectable at25min pi with a copy number of 87 per dish and theNP mRNA was detectable at 30min pi with copy numbersestimated at 05 per cell (Figure 3) results meeting therequirement for NDV infection and spread Interestinglytrace viral cRNA was detected after 5min of incubationand then detected when incubated for more than 25min incomparison no viral mRNA was detected with incubationsless than 30minThis result appears to contradict reports thatviral mRNA synthesis precedes cRNA synthesis in the earlyphase of infection [30ndash32 41] However these reports werebased on data detected after incubation In this study theresults displayed the unstable state of nascent viral RNA atthe early times of infection

Based on our results viral cRNA and mRNA can hardlybe detected in the initial 25min after virus-cell contact(Figure 3) It is reasonable to infer that those viral RNAs weredegraded at the every early time of infection similar to thatof influenza virus cRNA and mRNA [32] First of all viralmRNA will be degraded via basic mRNA decay machineryor antiviral mRNA turnover system [42ndash44] Secondly nakedgRNA or cRNA chain will be degraded by cellular nucleasesunless covered by NP proteins following the rule of six [1024] In addition NDV replication and transcription dependon the encapsidation of gRNA and cRNA Therefore NPproteins should be preferentially synthesized and NP gene

mRNA transcripts should be abundantly generated at theearliest period of infection [36] Viral cRNA and mRNAwould be steadily detected until their synthesis rates arehigher than degradation rates

After an incubation of 25min the gRNA level increasedrapidly During the first 6 hpi the levels of gRNA cRNA andmRNAwere always in a phase of exponential increase poten-tially related to the acute infection characteristics of NDVThe kinetics of NDV gRNA cRNA and mRNA synthesisin the infected DF-1 cells were shown to be consistent andcould be divided into three phases between 1 and 8 hpi whenviral RNAs accumulated exponentially (Phase I) followed bya plateau (Phase II) followed by a decrease between 16 and20 hpi (Phase III) Neither NDV transcription nor replicationwas predominant in the earliest phase of NDV infection

The replication of NDV involves the synthesis of gRNAand cRNA In this study cRNA was synthesized faster thangRNA (Figure 4) Our results showed that at 1 hpi cRNAsynthesis was initiated and its amount rapidly exceeding thatof gRNAThe cRNA levels were maintained 3ndash5 times higherthan that of gRNA until 48 hpi In other words the amountof cRNA was higher than that of gRNA altogether withininfected cells and supernatant Theoretically the amount ofgRNA would be higher than that of cRNA because onlygRNA (a template for mRNA and cRNA) is assembledinto viral particles and released out of the cells The rapidaccumulation of cRNA versus gRNA could be a specificcharacteristic of NDV or paramyxoviruses Similar resultswere reported in the Nipah virus [32] where the amountof intracellular vRNA of Nipah was much higher thanextracellular vRNA It was believed that Nipah virus particleswere not immediately released into the cell culture mediumHowever the proportion of cRNA in the intracellular vRNA


was not determined in the study Our results here suggest thatcRNA can be even higher than vRNA altogether in infectedcells and supernatant

To understand the above results the outcome of gRNAin viral particles released in supernatant should be recon-sidered Most gRNAs are assembled into viral particlesthat bud into cultural supernatant However those progenyviruses in supernatant actually gradually degrade under anenvironment temperature of 37∘C unless they infect cellsagain [45ndash48] The gRNA also decays following the deathof viral particles In contrast cRNA may behave differentlyAs a paramyxovirus NDV cRNA is totally encapsidated bythe NP protein that protects cRNA from degradation bycellular nucleases It is reasonable to infer that most cRNAis preserved in cells and accumulates steadily To concludea partially degradation of gRNA and preservation of cRNAwould explain why the amount of progeny cRNA was higherthan that of gRNA in our study

5 Conclusion

To conclude a real-time RT-PCR assay for absolute quan-titation of specific viral RNA fragments in La Sota infectedcells was developed for the first time With this method werevealed that the cRNA of NDV accumulated faster thanits vRNA The development of this assay will be helpful forfurther studies on the pathogenesis and control strategies ofNDV

Abbreviations

119862119902 Quantitation cycle

cRNA Antigenomic RNACV Coefficient of variationF Fusion proteingRNA Genomic RNAHN Haemagglutinin-neuraminidaseL Large proteinM Matrix proteinMOI Multiplicity of infectionmRNA Messenger RNAND Newcastle diseaseNDV Newcastle disease virusNP NucleoproteinP PhosphoproteinPBS Phosphate-buffered salinepi postinfectionqPCR Quantitative PCRRT Reverse transcriptionRT-PCR Reverse transcription polymerase chain reactionSPF Specific pathogen-freeTCID

50 50 tissue culture infection dose

vRdRp Viral RNA-dependent RNA polymerasevRNA Viral RNA

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Xusheng Qiu and Chan Ding designed the experiment anddrafted the paper Yang Yu Yuan Zhan and Nana Wei con-tributed to the establishment of the method Shengqing YuLei Tan and Cuiping Song made substantial contributions tothe experimental design All authors have read and approvedthe final paper

Acknowledgments

This work was funded through the Chinese National High-tech RampD Program (863 Program 2011AA10A209) theChinese Special Fund for Agroscientific Research in thePublic Interest (201303033) and the National Natural ScienceFoundation of China (31101822)

References

[1] Y M Saif and H J Barnes ldquoDiseases of poultryrdquo in NewcastleDisease Other Avian Paramyxoviruses and Pneumovirus Infec-tions Y M Saif H J Barnes J R Glisson A M Fadly LR McDougald and D E Swayne Eds Blackwell PublishingAmes Iowa USA 2008

[2] P J Miller E L Decanini and C L Afonso ldquoNewcastle diseaseevolution of genotypes and the related diagnostic challengesrdquoInfection Genetics and Evolution vol 10 no 1 pp 26ndash35 2010

[3] M A Mayo ldquoA summary of taxonomic changes recentlyapproved by ICTVrdquoArchives of Virology vol 147 no 8 pp 1655ndash1656 2002

[4] P J Miller L M Kim H S Ip and C L Afonso ldquoEvolutionarydynamics of Newcastle disease virusrdquo Virology vol 391 no 1pp 64ndash72 2009

[5] A M Linde M Munir S Zohari et al ldquoComplete genomecharacterisation of a Newcastle disease virus isolated during anoutbreak in Sweden in 1997rdquo Virus Genes vol 41 no 2 pp 165ndash173 2010

[6] A Wilde C McQuain and T Morrison ldquoIdentification of thesequence content of four polycistronic transcripts synthesizedin Newcastle disease virus infected cellsrdquo Virus Research vol 5no 1 pp 77ndash95 1986

[7] K Yusoff ldquoNewcastle disease virus Macromolecules andopportunitiesrdquo Avian Pathology vol 30 no 5 pp 439ndash4552001

[8] A C R Samson L Levesley and P H Russell ldquoThe 36Kpolypeptide synthesized in Newcastle disease virus-infectedcells possesses properties predicted for the hypothesized ldquoVrdquoproteinrdquo Journal of General Virology vol 72 no 7 pp 1709ndash17131991

[9] M Steward A C Samson W Errington and P T EmmersonldquoThe Newcastle disease virus V protein binds zincrdquo Archives ofVirology vol 140 no 7 pp 1321ndash1328 1995

[10] D Kolakofsky L Roux D Garcin and R W H RuigrokldquoParamyxovirus mRNA editing the ldquorule of sixrdquo and errorcatastrophe a hypothesisrdquo Journal of General Virology vol 86no 7 pp 1869ndash1877 2005

[11] S N Rout and S K Samal ldquoThe large polymerase proteinis associated with the virulence of Newcastle disease virusrdquoJournal of Virology vol 82 no 16 pp 7828ndash7836 2008


[12] M G Wise H S Sellers R Alvarez and B S Seal ldquoRNA-dependent RNA polymerase gene analysis of worldwide New-castle disease virus isolates representing different virulencetypes and their phylogenetic relationship with other membersof the paramyxoviridaerdquo Virus Research vol 104 no 1 pp 71ndash80 2004

[13] K Houben D Marion N Tarbouriech R W H Ruigrokand L Blanchard ldquoInteraction of the C-terminal domains ofsendai virus N and P proteins comparison of polymerase-nucleocapsid interactions within the paramyxovirus familyrdquoJournal of Virology vol 81 no 13 pp 6807ndash6816 2007

[14] D Kolakofsky P LeMercier F Iseni andD Garcin ldquoViral RNApolymerase scanning and the gymnastics of Sendai virus RNAsynthesisrdquo Virology vol 318 no 2 pp 463ndash473 2004

[15] T Ogino and A K Banerjee ldquoUnconventional mechanism ofmRNA capping by the RNA-dependent RNA polymerase ofvesicular stomatitis virusrdquoMolecular Cell vol 25 no 1 pp 85ndash97 2007

[16] A K Gupta M Mathur and A K Banerjee ldquoUnique cappingactivity of the recombinant RNA polymerase (L) of vesicularstomatitis virus association of cellular capping enzymewith theL proteinrdquo Biochemical and Biophysical Research Communica-tions vol 293 no 1 pp 264ndash268 2002

[17] A M Murphy M Moerdyk-Schauwecker A Mushegian andV Z Grdzelishvili ldquoSequence-function analysis of the Sendaivirus L protein domain VIrdquo Virology vol 405 no 2 pp 370ndash382 2010

[18] S M Horikami J Curran D Kolakofsky and S A MoyerldquoComplexes of Sendai virus NP-P and P-L proteins are requiredfor defective interfering particle genome replication in vitrordquoJournal of Virology vol 66 no 8 pp 4901ndash4908 1992

[19] M Hamaguchi K Nishikawa T Toyoda T Yoshida THanaichi and Y Nagai ldquoTranscriptive complex of newcastledisease virus II Structural and functional assembly associatedwith the cytoskeletal frameworkrdquo Virology vol 147 no 2 pp295ndash308 1985

[20] M Hamaguchi T Yoshida K Nishikawa H Naruse and YNagai ldquoTranscriptive complex of Newcastle disease virus IBoth L and P proteins are required to constitute an activecomplexrdquo Virology vol 128 no 1 pp 105ndash117 1983

[21] O S de Leeuw G Koch L Hartog N Ravenshorst and B PH Peeters ldquoVirulence of Newcastle disease virus is determinedby the cleavage site of the fusion protein and by both the stemregion and globular head of the haemagglutinin-neuraminidaseproteinrdquo Journal of General Virology vol 86 no 6 pp 1759ndash1769 2005

[22] T S Jardetzky and R A Lamb ldquoActivation of paramyxovirusmembrane fusion and virus entryrdquoCurrent Opinion in Virologyvol 5 pp 24ndash33 2014

[23] E C Smith A Popa A Chang C Masante and R EDutch ldquoViral entry mechanisms the increasing diversity ofparamyxovirus entryrdquo The FEBS Journal vol 276 no 24 pp7217ndash7227 2009

[24] R J Phillips A C R Samson and P T Emmerson ldquoNucleotidesequence of the 5rsquo-terminus of Newcastle disease virus andassembly of the complete genomic sequence agreement withthe lsquorule of sixrsquordquo Archives of Virology vol 143 no 10 pp 1993ndash2002 1998

[25] J A Bruenn ldquoA structural and primary sequence comparisonof the viral RNA-dependent RNA polymerasesrdquo Nucleic AcidsResearch vol 31 no 7 pp 1821ndash1829 2003

[26] D Kolakofsky T Pelet D Garcin S Hausmann J Curran andL Roux ldquoParamyxovirus RNA synthesis and the requirementfor hexamer genome length the rule of six revisitedrdquo Journal ofVirology vol 72 no 2 pp 891ndash899 1998

[27] S M Horikami S Smallwood and S A Moyer ldquoThe Sendaivirus V protein interacts with the NP protein to regulate viralgenome RNA replicationrdquoVirology vol 222 no 2 pp 383ndash3901996

[28] R A Lamb and D Kolakofsky ldquoFundamental virologyrdquo inParamyxoviridaeTheViruses andTheir Replication B B FieldsM D Kniepe and P M Howley Eds p 11 1395 LippincottWilliams ampWilkins Philadelphia Pa USA 2002

[29] B Tarus C Chevalier C Richard B Delmas C Primoand A Slama-Schwok ldquoMolecular dynamics studies of thenucleoprotein of influenza a virus role of the protein flexibilityin RNA bindingrdquo PLoS ONE vol 7 no 1 Article ID e300382012

[30] S PlumetW PDuprex andDGerlier ldquoDynamics of viral RNAsynthesis during measles virus infectionrdquo Journal of Virologyvol 79 no 11 pp 6900ndash6908 2005

[31] F S Heldt T Frensing and U Reichl ldquoModeling the intracel-lular dynamics of influenza virus replication to understand thecontrol of viral RNA synthesisrdquo Journal of Virology vol 86 no15 pp 7806ndash7817 2012

[32] L Chang A R Mohd Ali S S Hassan and S AbuBakarldquoQuantitative estimation of Nipah virus replication kinetics invitrordquo Virology Journal vol 3 article 47 2006

[33] S A Bustin J F Beaulieu J Huggett et al ldquoMIQE precispractical implementation of minimum standard guidelines forfluorescence-based quantitative real-time PCR experimentsrdquoBMCMolecular Biology vol 11 article no 74 2010

[34] S A Bustin V Benes J A Garson et al ldquoTheMIQE guidelinesminimum information for publication of quantitative real-timePCRexperimentsrdquoClinical Chemistry vol 55 no 4 pp 611ndash6222009

[35] X Wang Z Gong L Zhao et al ldquoComplete genome sequencesof newcastle disease virus strains isolated from three differentpoultry species in chinardquo Genome Announcements vol 1 no 4pp e00198ndashe00112 2013

[36] B P H Peeters Y K Gruijthuijsen O S de Leeuw and AL J Gielkens ldquoGenome replication of Newcastle disease virusinvolvement of the rule-of-sixrdquoArchives of Virology vol 145 no9 pp 1829ndash1845 2000

[37] A Dicarlo N Biedenkopf B Hartlieb A Kluszligmeier andS Becker ldquoPhosphorylation of marburg virus NP region IImodulates viral RNA synthesisrdquo Journal of Infectious Diseasesvol 204 supplement 3 pp S927ndashS933 2011

[38] M Iwasaki M Takeda Y Shirogane Y Nakatsu T Nakamuraand Y Yanagi ldquoThe matrix protein of measles virus regulatesviral RNA synthesis and assembly by interacting with thenucleocapsid proteinrdquo Journal of Virology vol 83 no 20 pp10374ndash10383 2009

[39] J Curran ldquoA role for the Sendai virus P protein trimer in RNAsynthesisrdquo Journal of Virology vol 72 no 5 pp 4274ndash42801998

[40] T L Liao C Y Wu W C Su K S Jeng and M M C LaildquoUbiquitination and deubiquitination of NP protein regulatesinfluenza A virus RNA replicationrdquo EMBO Journal vol 29 no22 pp 3879ndash3890 2010

[41] D Vester A Lagoda D Hoffmann et al ldquoReal-time RT-qPCRassay for the analysis of human influenza A virus transcription


and replication dynamicsrdquo Journal of Virological Methods vol168 no 1-2 pp 63ndash71 2010

[42] R Parker and H Song ldquoThe enzymes and control of eukaryoticmRNA turnoverrdquo Nature Structural and Molecular Biology vol11 no 2 pp 121ndash127 2004

[43] S Meyer C Temme and E Wahle ldquoMessenger RNA turnoverin eukaryotes pathways and enzymesrdquo Critical Reviews inBiochemistry and Molecular Biology vol 39 no 4 pp 197ndash2162004

[44] J Houseley and D Tollervey ldquoThe many pathways of RNAdegradationrdquo Cell vol 136 no 4 pp 763ndash776 2009

[45] P NWambura J Meers and P B Spradbrow ldquoThermostabilityprofile of Newcastle disease virus (strain I-2) following serialpassages without heat selectionrdquo Tropical Animal Health andProduction vol 38 no 7-8 pp 527ndash531 2006

[46] D J King ldquoSelection of thermostable newcastle disease virusprogeny from reference and vaccine strainsrdquoAvianDiseases vol45 no 2 pp 512ndash516 2001

[47] B Lomniczi ldquoThermostability ofNewcastle disease virus strainsof different virulencerdquo Archives of Virology vol 47 no 3 pp249ndash255 1975

[48] R E Gough ldquoThermostability of Newcastle disease virus inliquid whole eggrdquo Veterinary Record vol 93 no 24 pp 632ndash633 1973


The (minus) viral genomic RNA termed gRNA is a template forsynthesis of mRNA and the intermediate (+) viral genomicRNA (termed cRNA in this study) the latter was used as thetemplate for gRNA synthesis [7 26 27] The cRNAs serve asreplicative intermediates for gRNA duplication and are notknown to encode viral proteins [28]

The amounts of viral gRNA cRNA and mRNA report-edly change in a regular pattern in cells infected with themeasles virus influenza virus and Nipah virus [29ndash32]suggesting these RNA viruses regulate viral replication andtranscription through specific molecular mechanisms Inthis study a strand-specific real-time RT-PCR assay wasdeveloped to distinguish viral gRNA cRNA and mRNAlevels in NDV-infected cells This will aid studies into themolecular mechanisms of viral replication and transcriptionviral pathogenesis and control strategies for NDV

2 Materials and Methods

21 Cell and Virus DF-1 cells were obtained from the Amer-ican Type Culture Collection (ATCC Manassas VA USA)and grown in RPMI-1640 media (10 fetal bovine serum2mM L-glutamine 100 IUmL penicillin 100 120583gmL strepto-mycin and 10mM HEPES buffer) at 37∘C and 5 CO

2 Cell

culture reagents were obtained from Gibco (Rockville MDUSA)

NDVLa Sota strainwas obtained from theChina Instituteof Veterinary Drug Control (Beijing China) and propagatedin 9- to 11-day-old specific-pathogen-free (SPF) embryosTheinfected allantoic fluid was harvested between 84 and 120 hThe virus was determined for the 50 tissue culture infectivedose (TCID

50) and kept at minus80∘C before use

22 Virus Infection The incubation time for the virus infec-tion was optimized first DF1 cells (3 times 105 cells) werecultivated in 60mm dishes at 37∘C and 5 CO

2 For La

Sota infection a monolayer of DF1 cells was washed withphosphate-buffered saline (PBS) three times and 1mL ofserum-free 1640 medium containing NDV La Sota at amultiplicity of infection (MOI) of 001 was then added At5 s 1min 5min 10min 15min 20min 25min 30min and60min postinfection (pi) the cells were washed again withPBS to remove unbound virus particles and harvested forRNA extraction and subjected to real-time RT-PCR Theoptimal incubation time for NDV infection was based onwhen the cDNAs of gRNA cRNA and mRNA of NP startedto stably accumulate The quantitation cycle (119862

119902) values of

each RNA at this time point were considered residual viralRNAs after incubation and set as baseline values Accordingto the optimized incubation time determined DF1 cells wereinfectedwith La Sota at anMOI of 001 After incubation cellswere washed with PBS and then grown in 4mL 1640mediumcontaining 1 (vv) fetal calf serum at 37∘C and 5 CO

2 The

cells and supernatant were harvested at different time pointsfor the assay

23 RNA Extraction and Strand-Specific Reverse Transcrip-tion After infection with NDV La Sota the supernatant and

cells were collected and thoroughly mixed and 500120583L ofthe mixture was lysed with 1mL TRIZol reagent (Invitro-gen Carlsbad CA USA) The total RNA of the virus wasextracted according to the manufacturerrsquos instructions TheRNA pellets were resuspended in 50 120583L RNase-free waterafter washing with 70 ethanol To degrade cellular DNADNase I was added to the samples at a final concentrationof 1 U120583L at 37∘C for 30min and then inactivated at 65∘C for10min in the presence of 5mM EDTA

Strand-specific reverse transcription of NDV gRNAcRNA and mRNA was performed with specifically designedreverse transcription primer sets (Figure 1) To quantify thethree kinds of cDNAs with one pair of primers reverse tran-scription primers specific for gRNA cRNA or mRNA weredesigned using the Lasergene software suite (DNASTARMadison WI USA)

The sequence of the gRNA-specific reverse transcriptionprimer PLa-Gwas 51015840-ACGATAAAAGGCGAAGGAGCA-31015840 (genomic position 22ndash44 nt) which initiated synthesis ofthe unique leader sequence of the NDV genome

The sequence of primer PLa-R specific to the P gene ofviral cRNA was 51015840-GTC GTT TGC TCG GGT GTG GAT G-31015840 (genomic position 2 119-2 139 nt)

The extracted mRNA segments involving viral mRNAwere reverse-transcribed byPLa-18V (51015840-TTTTTTTTTTTTTTT TTT AGC-31015840)

Each 10 120583L of the above RNA samples was reverse-transcribed by respective primers in the presence of M-MLV reverse transcriptase (Promega Madison WI USA)according to the protocol Briefly 200 U M-MLV reversetranscriptase 5120583L 5 times RT buffer 10 120583L RNA sample 10mmolspecific RT primer 10mmol dNTPs and ultrapure water(Gibco BRL Gaithersburg MD USA) were added to a finalvolume of 25 120583L and mixed The mixture was incubated at42∘C for 60min and then 75∘C for 15min to denature reversetranscriptase The reverse transcripts were precipitated usingtwo volumes of ethanol Finally the yielded pellet wasdissolved in 20120583L of ultrapure water (Gibco BRL) and keptat minus70∘C until use

24 Real-Time Quantitative Polymerase ChainReaction (PCR) Assay

241 Construction of Plasmid T-NP as a Standard for theAssay To construct a standard curve for the linear corre-lation between 119862

119902values and molecular numbers of tar-

get cDNAs plasmid T-NP was constructed and used as astandard Genomic RNA was reverse-transcribed by randomhexamer primers and 5 120583L of the resultant cDNA fragmentswas then added into a 50120583L PCR reaction system containing25U TaKaRa LA Taq 5 120583L 10 times LA PCR Buffer II (Mg2+Plus) 1120583L 20120583M former primer PNPF 1 120583L 20120583M reversetranscription- (RT-) primer PNPR and 8 120583L 25mM dNTPmixtureThe primer pairs of PNPF (51015840-CAG TGA TGACCCAGA AGA TAG ATG-31015840) and PNPR (51015840-CGC AAA GCTCAT CTG GTC ACT ATC-31015840) were used to amplify the NPgene The PCR cycle conditions were 95∘C 1min 25 cycles of94∘C 30 s 55∘C 30 s and 72∘C 90 s followed by 72∘C 10min


+++++++++++++++

+++++++++++++

++++++++++

+++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++


Cap

Cap

Cap

Cap

Cap

Cap


3998400

5998400 3998400



M

mRNA

P mRNA

F

mRNA

HN

mRNA

LmRNA

NP

mRNAminusAn

minusAn

minusAn

minusAn

minusAn

minusAn

(a)

463 607

Le NP

Le NP

NP

P

P

C

gRNA

mRNA

cRNA

Pla-18T

Pla-G

Pla-R

Pla-rt13

Pla-rt14

Pla-rt13

Pla-rt14

Pla-rt14

Pla-rt13

Poly(A)n

3998400

5998400

39984005998400

3998400

5998400

(b)




















3 Results






119902value


119902



119902values







4 Discussion




9492908886848280787674727068666462


minusdIdT

()

minus5

minus15

95

85

75

65

55

45

35

25

15

5

(a)

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10

y = minus32901x + 38922

R2 = 09982

Thre

shol

d cy

cle (C

T)



(b)


00

05

10

15

20

25

gRNAcRNA

mRNA



log10

copi

esc

ell

5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m









gRNAcRNA

Hours postinfection


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)











5 Conclusion


Abbreviations









Acknowledgments


References





















































+++++++++++++++

+++++++++++++

++++++++++

+++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++


Cap

Cap

Cap

Cap

Cap

Cap


3998400

5998400 3998400



M

mRNA

P mRNA

F

mRNA

HN

mRNA

LmRNA

NP

mRNAminusAn

minusAn

minusAn

minusAn

minusAn

minusAn

(a)

463 607

Le NP

Le NP

NP

P

P

C

gRNA

mRNA

cRNA

Pla-18T

Pla-G

Pla-R

Pla-rt13

Pla-rt14

Pla-rt13

Pla-rt14

Pla-rt14

Pla-rt13

Poly(A)n

3998400

5998400

39984005998400

3998400

5998400

(b)




















3 Results






119902value


119902



119902values







4 Discussion




9492908886848280787674727068666462


minusdIdT

()

minus5

minus15

95

85

75

65

55

45

35

25

15

5

(a)

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10

y = minus32901x + 38922

R2 = 09982

Thre

shol

d cy

cle (C

T)



(b)


00

05

10

15

20

25

gRNAcRNA

mRNA



log10

copi

esc

ell

5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m









gRNAcRNA

Hours postinfection


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)











5 Conclusion


Abbreviations









Acknowledgments


References

































































3 Results






119902value


119902



119902values







4 Discussion




9492908886848280787674727068666462


minusdIdT

()

minus5

minus15

95

85

75

65

55

45

35

25

15

5

(a)

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10

y = minus32901x + 38922

R2 = 09982

Thre

shol

d cy

cle (C

T)



(b)


00

05

10

15

20

25

gRNAcRNA

mRNA



log10

copi

esc

ell

5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m









gRNAcRNA

Hours postinfection


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)











5 Conclusion


Abbreviations









Acknowledgments


References
























































119902value


119902



119902values







4 Discussion




9492908886848280787674727068666462


minusdIdT

()

minus5

minus15

95

85

75

65

55

45

35

25

15

5

(a)

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10

y = minus32901x + 38922

R2 = 09982

Thre

shol

d cy

cle (C

T)



(b)


00

05

10

15

20

25

gRNAcRNA

mRNA



log10

copi

esc

ell

5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m









gRNAcRNA

Hours postinfection


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)











5 Conclusion


Abbreviations









Acknowledgments


References





















































9492908886848280787674727068666462


minusdIdT

()

minus5

minus15

95

85

75

65

55

45

35

25

15

5

(a)

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10

y = minus32901x + 38922

R2 = 09982

Thre

shol

d cy

cle (C

T)



(b)


00

05

10

15

20

25

gRNAcRNA

mRNA



log10

copi

esc

ell

5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m 5s

1m

5m

10m

15m

20m

25m

30

m60

m









gRNAcRNA

Hours postinfection


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)











5 Conclusion


Abbreviations









Acknowledgments


References





















































gRNAcRNA

Hours postinfection


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

(a)

mRNA


9

10

11

12

0 4 8 12 16 20 24 28 32 36 40 44 48

log10

copi

esd

ish

Hours postinfection

(b)











5 Conclusion


Abbreviations









Acknowledgments


References























































5 Conclusion


Abbreviations









Acknowledgments


References




















































development of strand-specific real-time rt-pcr to

Documents