Journal of Virological Methods 131 (2006) 122–129

Use of quantitative real-time PCR (qRT-PCR) to measure cytokinetranscription and viral load in murine cytomegalovirus infection�

Yajarayma J. Tang-Feldmana,b,∗, Angela Wojtowicza,b, G. Raymond Lochheada,b,Merica A. Halea,b, Yueju Lic, Claire Pomeroya,b,d

a Department of Internal Medicine, Division of Infectious and Immunologic Diseases, University of California,Davis Health System, Sacramento, CA, USA

b Department of Medical Microbiology and Immunology, University of California, Davis, Davis, Sacramento, CA, USAc Department of Public Health, School of Medicine, University of California, Davis, Davis, CA, USAd Northern California Veterans Affairs Health Care System, Mather Branch, Sacramento, CA, USA

Received 7 June 2005; received in revised form 25 July 2005; accepted 26 July 2005Available online 2 September 2005

Abstract

ub-clinicala mediatee Increasedm fection.TI ioni d in anyo llyi respondingt cytokiner ersusc onses tot©

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A quantitative real-time PCR (qRT-PCR) assay was developed to measure cytokine transcription profiles and viral load during snd clinical infection with murine cytomegalovirus (MCMV). Primers/fluorogenic probes specific for mouse cytokines and for the imarly gene 1 (IE1) of MCMV were used to quantitate cytokine responses and viral load in various organs of MCMV infected mice.RNA levels of TNF-�, INF-� and IL-10 were detected in the spleens, lungs and livers of clinically infected mice at 5 days post-inranscription of these cytokines was 2–5-fold lower (p = 0.07 for each cytokine) in the spleens and 10–100-fold lower in the lungs (p = 0.03 for

NF�, not significant for IL-10 and TNF�) and livers (p < 0.05 for each cytokine) of sub-clinically infected mice. Clinical MCMV infectnduced high levels of IL-6 in the lungs and spleens of infected animals, while no significant transcription of IL-6 was detectergan during sub-clinical infection (p < 0.05). The timing of peak amounts of INF-�, IL-10 and IL-6 observed in the spleens of clinica

nfected mice correlated with high viral loads in these organs. Cytokine expression rose in the salivary glands later, at day 15, coro the increase in salivary gland viral load. The qRT-PCR demonstrates that infection with MCMV induces an organ-specificesponse characterized by the production of TNF-�, INF-�, IL-6 and IL-10 which correlates with severity of the disease (sub-clinical vlinical) and with viral load. In summary, qRT-PCR is a sensitive and accurate method to study MCMV infection and host resphe virus.

2005 Elsevier B.V. All rights reserved.

eywords: MCMV; Quantitative RT-PCR; Cytokine response; Viral load

. Introduction

Cytomegalovirus (CMV) infection is a major cause oforbidity and mortality in immunocompromised patients,specially those with HIV/AIDS infection and in bone mar-

� This work was presented in part at the 42nd Annual Meeting of thenfectious Diseases Society of America, Boston, MA, USA, 2004.∗ Corresponding author. Present address: University of California, Davisealth System, 4150 V Street, Suite 1100, Sacramento, CA, USA. Tel.: +116 734 3578; fax: +1 916 734 7055.

E-mail addresses: yjtangfeldman@ucdavis.edu (Y.J. Tang-Feldman),laire.pomeroy@ucdmc.ucdavis.edu (C. Pomeroy).

row and solid organ transplant recipients (Ho, 1995). CMV isalso the most common infectious cause of congenital mamations (Stagno et al., 1986). CMV infection is most commonly acquired during childhood, as reflected by seroptivity prevalence rates of 40–95% in adults around the wIndeed, most CMV infections are sub-clinical and asytomatic, and therefore go unrecognized.

Murine (mouse) models of CMV (MCMV) infection auseful for studying host response to the virus because mathe immune responses parallel those of humans. The mmodel is an excellent tool to study the course of both clinand sub-clinical infection, as well as viral latency and re

166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2005.07.013

Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122–129 123

tivation. Research on MCMV has provided significant infor-mation on the pathogenesis of clinical CMV disease, includ-ing the role played by cytokines in host defense (Orange andBiron, 1996a; Pavic et al., 1993; Pomeroy et al., 1991).

Cytokines are critical in the defense against MCMV. TNF-� has been reported to limit titers of MCMV in organs in vivo,and has been associated with protective effects during acuteinfection in adult BALB/c mice (Orange and Biron, 1996b;Pavic et al., 1993). However, some studies have shown thatneutralization of TNF-� in MCMV infected mice had noeffect on viral replication or disease outcome (Shanley etal., 1994). INF-� has been associated with antiviral activitiesduring MCMV infection in both BALB/c and C57BL/6 mice(Orange and Biron, 1996b). Administration of high doses ofINF-� increased morbidity and mortality in BALB/c micesuggesting that the cytokine has both beneficial and detri-mental effects on the host (Pomeroy et al., 1998). IL-6, IL-4and IL-10 have also been reported to play a role in MCMVinfection (Geist and Hinde, 2001).

Studies of cytokine production in response to MCMVinfection have usually assessed serum levels or systemiccytokine response in clinical, symptomatic infections usingELISA based assays. Others have used RNA protectionassays or reverse transcriptase PCR to study cytokine expres-sion in bronchoalveolar lavage and spleen (Karupiah et al.,1998; Wu et al., 2001). Little is known about organ-specificc tol

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All mice were housed in AAALAC-approved SPF facilitiesat our institution and had free access to food and water. Ani-mal studies and protocols were approved by the UC DavisInstitutional Animal Care and Use Committee (IACUC).

2.2. Virus and infection

MCMV Smith strain was obtained from American TypeCulture Collection (ATCC) (Manassas, VA). The virus wasmaintained by salivary gland passage in BALB/c mice, anda salivary gland homogenate (SGV) was prepared in RPMI(Gibco Laboratories, Grand Island, NY) as described else-where (Pomeroy et al., 1991). To determine the sub-lethaldose to establish a clinical infection, a LD50 of the SGV wasperformed using standard protocols (Pomeroy et al., 1991).Mice were infected by intraperitoneal (i.p.) injection of 0.2 mlof a sub-lethal dose (a dose of virus appropriately adjusted tocause severe disease but not lethal infection,∼104 PFU) ofthe SGV in RPMI. This dose was 2-fold lower than the LD50dose. Clinically infected mice were observed and scored dailyfor signs: lethargy, weight loss, ruffled fur, hunched postureand squinty eyes. Groups of mice (n = 8) were sacrificed atdays 5 and 15 post-infection for analysis of cytokine expres-sion and viral load. To establish a sub-clinical infection, micereceived by i.p. route 0.2 ml of a sub-clinical dose of theSGV (∼300 PFU), an amount previously found to infectm ta).S and1 nin-f andh pres-s

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ytokine response during MCMV infection, primarily dueimitations of the techniques available.

In the past 10 years, quantitative real-time PCR (qCR) has become one of the techniques of choice toene expression in cells and tissues. The use of fluore

abeled probes increases the sensitivity and specificity oystem allowing detection of very minute amounts of tcripts. This method has been used to study cytokine reso M. bovis in guinea pigs (Kawahara et al., 2002), cytokinend chemokine transcription in response toH. influenzae inumans (Tong et al., 2001), and to assess cytokine mRNA lels in feline monocytes (Kipar et al., 2001). qRT-PCR assayave also been used to quantitate viral loads in a numbiral infections including CMV (Wheat et al., 2003; Vlieget al., 2003; Gault et al., 2001; Jebbink et al., 2003).

In this study, a quantitative real-time PCR assay was dped to analyze the organ-specific transcription levelsanel of cytokines (IL-2, IL-6, IL-10, TNF-� and INF-�) inelected organs during sub-clinical/asymptomatic infecnd clinical infection with MCMV. These cytokine transcr

ion patterns were then correlated with the organ-specificoad.

. Materials and methods

.1. Mice

Pathogen-free 3–4 weeks old BALB/c female mice wurchased from Harlan Sprague Dawley (Indianapolis,

ice without causing any clinical signs (unpublished daub-clinically infected mice were sacrificed at days 55 post-infection. In each experiment, a control group (u

ected mice) received 0.2 ml of RPMI intraperitoneally,ad their organs removed and analyzed for cytokine exion, as in the infected groups.

Signs of MCMV infection usually appear 2 days after ccal infection and continue for about 5 more days with recry of the mice starting around days 8–10 post-infection.

ime points used in this study encompass the peak ofigns (5 days) to the complete resolution of clinically apnt signs (15 days).

.3. Tissue collection

At each time point, a group of mice (n = 8) was sacriced and samples of lungs, livers, spleens, salivary glandmall intestines removed and placed in Nucleic Acid Puation Lysis Solution (Applied Biosystems, Foster City, Cor DNA and RNA extraction.

.4. Nucleic acid extraction and cDNA synthesis

DNA was extracted from each organ with the DNeasyue Kit (Qiagen, Valencia, CA) following the manufacturenstructions. RNA extraction and cDNA synthesis wereormed at the Molecular Core Facility, School of Veterinedicine at our institution using the 6700 RNA extr

ion apparatus (Applied Biosystems) as per instructionsuidelines. Briefly, cDNA was synthesized using 100 u

124 Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122–129

of SuperScript II (Invitrogen, Carlsbad, CA), random hex-amer primers (300 ng), 10 U of RNaseOut (Invitrogen), 1 mMdNTPs and 20�l of the RNA preparation in a final volumeof 40�l. After 50 min of incubation at 42◦C, 10�l of waterwere added and the reaction was terminated by heating for5 min at 95◦C.

2.5. Real-time PCR of cytokines

Quantitative transcription profile of IL-2, IL-6, IL-10,TNF-� and INF-� in each organ was determined by qRT-PCR using the Assay on Demand kits (Applied Biosystems)with the respective primers/fluorogenic probe mix specific foreach mouse cytokine. In addition, transcription of the house-keeping genes, GAPDH and HPRT, in each organ was deter-mined using specific primer/fluorogenic probe mix (AppliedBiosystems). Real-time PCR reactions were set up in dupli-cates for each of the cytokines and housekeeping genes ineach organ analyzed. Amplification conditions were identi-cal for all reactions and consisted of: 2 min at 50◦C, 10 minat 95◦C, 40 cycles of 15 s at 95◦C and 60 s at 60◦C. Reactionsamples had a final volume of 6�l consisting of 3.5�l of Uni-versal Master mix containing the specific primer/probe mix(Applied Biosystems) and 2.5�l of the respective cDNA.Amplifications were run in an ABI Prism 7900 SequenceDetection System (Applied Biosystems).

hereue targetC ingg dif-f eachra sa d thec nor-mec or-m andrl calcu-l genei ctedm

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as per instructions. Samples were set up in 50�l final vol-umes containing 25�l of the master mix, 30 pmoles of therespective forward and reverse primer, and the reaction wasmade up to 50�l with sterile water. PCR conditions includeda hot start step at 95◦C for 10 min, followed by 40 cycles con-sisting of 95◦C for 45 s, 55◦C for 45 s and 72◦C for 1 minwith a final extension at 72◦C for 7 min.

The respective amplicon was cloned into a pCR®II TOPOvector using the TOPO TA Cloning kit (Invitrogen, USA) asper manufacturer’s instructions. Plasmids were purified usingthe Qiagen Plasmid Mini Prep (Qiagen) and quantificationwas done by spectrophotometry.

2.7. Quantification of MCMV using real-time PCR

Serial 10-fold dilutions of the plasmid containing the IE1insert were done in calf-thymus DNA (30 ng/ml) (Sigma,St. Louis, MO) and used to construct a standard curve.The concentrations of the plasmid dilutions ranged from2 to 2× 109 copies. The forward and reverse primerslisted above and the TaqMan probe were designed usingthe Primer Express Software (Applied Biosystems). Thesequence of the TaqMan probe was 5′-TTCTCTGTCA-GCTAGCCAATGATATCTTCGAGC-3′. For real-time PCR,the probe was labeled at the 5′ end with the reporter dye FAMa ′ eP Mix( s in1 gt oft -t1 t6 ver-a latem ofl -b ationso ed as

Final quantification was performed as described elsewsing the comparativeCT method (Kipar et al., 2001). Forach experimental sample, the difference between theT value and theCT value of the most stable housekeepene for each tissue type was used to normalize for

erences in the amount of total nucleic acid added toeaction and the efficiency of the RT step (�CT). For rel-tive quantification by the comparativeCT method, valuere then expressed relative to a reference sample callealibrator. The calibrator is the weakest signal from thealization (�CT) in each tissue type. The�CT for each

xperimental sample was subtracted from the�CT of thealibrator (��CT). The amount of target (linear value) nalized to an internal control or housekeeping gene

elative to the calibrator was determined by 2��CT. Theinear expression of each gene in each tissue was thenated by subtracting the linear expression value of eachn uninfected mice (baseline value) from that of the infe

ice.

.6. Amplification and cloning of MCMV immediatearly 1 (IE1) gene

DNA was extracted from MCMV Smith strain (ATCCsing the QIAmp DNA Blood Mini Kit (Qiagen) as per mafacturer’s instructions. Oligonucleotide primers usedmplification of the IE1 gene were: forward primer 5′-TCA-CCATCAACTCTGCTACCAAC-3′ and reverse primer 5′-TCTGAAACAGCCGTATATCATCTTG-3′. Amplificationas performed with the HotStarTaq Master Mix kit (Qiag

nd at the 3end with the quencher dye TAMRA. Real-timCR was performed using the TaqMan Universal Master

Applied Biosystems). Reactions were set up in triplicate2�l volumes consisting of 7�l of the cocktail containin

he Universal Master mix, 400 nM of each primer, 80 nMhe TaqMan probe and 5�l of the respective plasmid diluion. Amplification conditions consisted of 2 min at 50◦C,0 min at 95◦C, and 40 cycles of 15 s at 95◦C and 60 s a0◦C. A standard curve was constructed by plotting ageCT values against the logarithm of the target tempolecules obtained from the plasmid, followed by a sum

east squares regression analysis (Fig. 1). Target copy numers in tissue samples were calculated using the equbtained in regression analysis. Results were express

Fig. 1. Standard curve for MCMV DNA quantification (IE1 gene).

Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122–129 125

DNA copies/100 mg of tissue (Vliegen et al., 2003). SinceDNA yield per 100 mg of tissue differs from tissue to tissue,DNA was extracted from 100 mg of each tissue and analyzedusing the Qiagen DNeasy kit (Qiagen). All extractions weredone in quadruplicate and the average was used to determinethe DNA yield/100 mg of each tissue type. Because only1�g of DNA is used in qRT-PCR, the number of MCMVcopies/100 mg of tissue was considered to equal the numberof copies obtained in the real-time PCR reaction multipliedby the amount of total DNA obtained for each specific tissuetype.

2.8. Statistical analysis

To determine if the cytokine response during clinical infec-tion was significantly greater than during sub-clinical infec-tion, two-samplet-tests were used on double-log transformeddata, separately for each cytokine in each organ; 95% confi-dence intervals for the difference in means were calculatedon the double-log transformed scale and then transformedback to give confidence intervals in the original units of mea-surements. Similarly, cytokine response was compared foreach pair of time points usingt-tests and 95% confidenceintervals on the double-log scale. Pearson correlations werecalculated between cytokine and viral load. All data wereplotted to check normality and non-parametric alternativep rrela-t tionso ans-f allat

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Table 1Clinical signs of MCMV infection in BALB/c mice

Day post-infection

1 2 3 5 8 15

Weight lossa (%) 2.5 10.1 13.4 23.2 21.5 5.0

Signsb

Lethargic +1 +1 +2 +3 +2 0Ruffled fur +1 +1 +2 +3 +2 0Hunched posture +1 +1 +2 +3 +1 0Squinty eyes 0 +1 +2 +2 0 0

Score: 0, no signs observed; +1, slightly lethargic, ruffled primarily aroundneck, hunched while sitting; +2, lethargic, ruffled fur around neck area andtorso, back bone prominent when sitting, squinty eyes; +3, very lethargic,ruffled fur in most areas, red skin around neck area, hunched when walking,more prominent squinty eyes.

a Values of percent body weight loss are the average of the mice remainingin all groups at the respective time points.

b Signs and severity represent compiled observations of the mice remain-ing in all groups at the respective time points.

3.2. Quantification of cytokine transcription duringsub-clinical and clinical infection measured by qRT-PCR

Cytokine response in the various organs was assessed at5 and 15 days post-infection. Linear expression values ofcytokines in infected mice were standardized to the back-ground or baseline expression in uninfected mice. A sub-stantial variability in cytokine response in each organ wasobserved in individual animals. TNF-�, INF-� and IL-6 tran-scripts reached highest levels in the lungs and livers of clin-ically infected mice at days 5 post-infection (Table 2). Inthe spleens, levels of TNF-� and IL-6 rose at day 5 post-infection and remained elevated throughout the study period.Transcription of these cytokines at day 5 post-infection insub-clinically infected mice was 2–100-fold lower than inclinical infection. IL-6 transcription levels were also elevatedin the spleens and lungs of clinically infected mice, while nosignificant transcription of IL-6 was detected in any organsof the sub-clinically infected mice (Table 2).

In contrast to other organs, cytokine expression levels inthe salivary glands of sub-clinically infected mice did notincrease significantly at day 5 post-infection. Expression ofTNF-�, INF-� and IL-10 in salivary glands of clinicallyinfected mice was detected at day 5, remaining significantlyelevated above baseline at day 15 (p < 0.04) (Table 2). Lev-els of these cytokines in salivary glands of clinically infectedm tedm fs ithp irall es

alli timep

rocedures (Wilcoxon rank sum test, Spearman rank coion) were carried out if the data suggested possible violaf distribution assumptions even after log or double-log tr

ormation. All tests were two-sided at level 0.05, andnalyses were performed using SAS Version 8.2 (SAS Insti-

ute Inc., 1999–2001).

. Results

.1. Establishment of sub-clinical and clinical infectionith MCMV

Little is known about cytokine response during asyomatic MCMV infection. To determine the usefulnesshe qRT-PCR assay to study cytokine responses dub-clinical infection, two groups of mice were infectlinically infected mice received a sub-lethal dosehe MCMV-SGV (∼104 PFU) capable of causing seveisease but no lethal infection; sub-clinically infected meceived a dose of the MCMV-SGV (∼300 PFU) previouslound to cause infection but no apparent clinical sunpublished data). Clinically infected mice develoypical signs of the disease. Signs of MCMV infectppeared at day 1 post-infection, and peaked aroundafter infection. By day 10 post-infection most clinica

pparent signs subsided and by day 15 mice applinically recovered (Table 1). No clinical manifestatiof disease was observed in the sub-clinically infeice.

ice were 5–200-fold higher than in sub-clinically infecice at both day 5 and day 15 (Table 2). The severity o

igns observed in clinically infected mice correlated weak TNF-� and INF-� responses (day 5) and highest v

oads in the lungs and livers, and peak INF-� response in thpleens (day 5).

No cytokine mRNA transcripts were detected in the smntestines. IL-2 was not detected in any organ at theoints analyzed (data not shown).

126 Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122–129

Table 2TNF-�, INF-�, IL-6 and IL-10 transcription profile and viral load at 5 and 15 days post-sub-clinical and clinical infection with MCMV

Tissue Day post-infection Linear valueb Viral loadc Signsd

TNF-� (S.D.)a IL-6 (S.D.)a IL-10 (S.D.)a INF-� (S.D.)a

SpleenSub-clinical 5 612 (±1538) 1 (±1) 99 (±215) 1069 (±2700) 1.1× 106 −Clinical 5 2495 (±2100) 395 (±392) 571 (±440) 1744 (±2150) 4.8× 107 +p 0.07 0.003 0.08 0.16 0.036

Sub-clinical 15 217 (±238) 2 (±4) 109 (±90) 206 (±141) 3.9× 102 −Clinical 15 3716 (±5068) 2634 (±3370) 88 (±110) 411 (±595) 5.1× 106 −p 0.02 0.0015 0.91 0.98 0.007

LungSub-clinical 5 69 (±42) 0 (±3) 196 (±151) 27 (±18) 6.8× 102 −Clinical 5 3821 (±5300) 64 (±108) 832 (±1450) 350 (±443) 1.6× 105 +p 0.007 0.23 0.29 0.03 0.042

Sub-clinical 15 6 (±11) 5 (±6) 7 (±8) 1 (±2) 0 −Clinical 15 508 (±1200) 0 (±3) 933 (±1060) 102 (±148) 1.5× 102 −p 0.001 0.009 0.38 0.15 0.0001

LiverSub-clinical 5 25 (±32) 0 64 (±44) 6 (±10) 1.5× 103 −Clinical 5 5770 (±9374) 91 (±160) 924 (±1800) 600 (±1080) 7.5× 106 +p 0.007 0.002 0.03 0.01 0.012

Sub-clinical 15 65 (±59) 0 7 (±2) 5 (±2) 0 −Clinical 15 59 (±176) 24 (±11) 3 (±15) 8 (±21) 7.4× 103 −p 0.13 0.01 0.38 0.11 0.001

Salivary glandSub-clinical 5 10 (±76) 0 19 (±14) 7 (±7) 1.1× 102 −Clinical 5 2131 (±2068) 12 (±15) 630 (±1430) 95 (±192) 1.8× 105 +p 0.03 0.01 0.099 0.05 0.005

Sub-clinical 15 245 (±186) 0 25 (±28) 13 (±7) 1.6× 105 −Clinical 15 1128 (±1590) 6 (±8) 703 (±1013) 149 (±252) 1.0× 107 −p 0.007 0.04 0.001 0.003 0.08

p Values represent clinical vs. sub-clinical values.a S.D., standard deviation.b Linear value, linear value in infected mice− linear value in uninfected mice.c DNA copies/100 mg of tissue (IE-1).d (+), Signs observed (seeTable 1); (−), no symptoms observed.

3.3. Quantification of MCMV viral load in specificorgans during sub-clinical and clinical infection

In our experiments, none of the sub-clinically infectedmice showed signs of MCMV disease. Despite this, MCMVDNA was detected in the lungs, livers and spleens at day 5post-infection (Table 2). By day 15 post-infection, viral DNAhad considerably decreased in these organs. In contrast, sig-nificant viral DNA was detected in the salivary glands of sub-clinically infected mice at day 15 post-infection, althoughlevels remained three logs below those found in the salivaryglands of clinically infected animals (Table 2).

Viral load was 10–10,000-fold higher in most organsduring clinical infection compared to sub-clinical infection.DNA copies of MCMV in the lungs, livers and spleens ofclinically infected mice were highest at day 5 post-infectionand ranged from 105 to 107 DNA copies/100 mg of tissue(Table 2). By day 15, viral DNA had decreased by 1000-foldin lungs and livers of clinically infected mice, while viral load

in the spleens remained relatively high throughout the studyperiod in parallel with high levels of cytokines. In contrast, inthe salivary glands, a higher viral load was detected later, atday 15 post-infection (Table 2, Fig. 2). Viral DNA was also

Fig. 2. MCMV viral load in tissues from clinically infected mice.

Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122–129 127

Table 3Organ-specific correlations between viral load and cytokine response (Spearman rank correlation, significance,p = 0.05 or lower; trends,p < 0.10)

Organ IL-10 (p-value) IL-6 (p-value) INF-� (p-value) TNF-� (p-value)

Liver 0.52 (<0.001) 0.54 (<0.001) 0.47 (0.001) 0.49 (<0.001)Lung 0.28 (0.066) 0.15 0.20 0.43 (0.005)Salivary gland 0.53 (<0.001) 0.53 (<0.001) 0.62 (<0.001) 0.52 (<0.001)Small intestine 0.20 0.05 −0.12 0.21Spleen 0.41 (0.004) 0.42 (0.003) 0.54 (<0.001) 0.45 (0.002)

detected in the salivary glands of sub-clinically infected miceat 15 days after infection (Table 2).

3.4. Correlation of organ-specific cytokine transcriptionand viral load

Significant correlation was found between extent ofcytokine production and viral load in spleens (p < 0.005 forIL-6, IL-10, INF-� and TNF�), livers (p < 0.005 for IL-6,IL-10, INF-� and TNF-�), lungs (p = 0.005 for TNF-�) andsalivary glands (p < 0.001 for IL-6, IL-10, INF-� and TNF-�)(Table 3). Notably, cytokine levels peaked in lungs, and liversat day 5 when viral loads were higher, and persisted elevatedlonger in the spleens, where viral load remained elevatedlonger. In contrast, cytokine levels (IL-10 and INF-�) stayedlow in salivary glands early when viral replication was lim-ited, and rose later at day 15, in concert with the later increasesin viral load. Viral DNA copy numbers in small intestineswere 1000–10,000-fold lower than in the spleens, livers andlungs. The levels of cytokine transcription in these organswas 10–100-fold lower than in spleens, livers and lungs (datanot shown). These findings are consistent with organ-specificcytokine responses that parallel organ-specific differences inextent and timing of viral replication.

4

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MVi ngs,s F-T ruml tion

in BALB/c mice, probably due to an initial response medi-ated by natural killer (NK) cells (Orange et al., 1995; Shanleyet al., 1994). High levels of INF-� have also been detectedin the serum of infected mice up to 6–7 days after infection(Pomeroy et al., 1998).

Elevated levels of TNF-� were detected at day 5 duringclinical infection in all the organs analyzed. Transcriptionof TNF-� was markedly increased in the spleens, lungs andlivers during early stages of infection (day 5) and remainedelevated in the spleens throughout the study period in parallelwith a high viral load. These results are consistent with pre-vious reports of high levels of TNF-� in the serum of acutelyinfected mice, suggesting a role for TNF-� in the initial con-trol of the infection (Trgovcich et al., 2000; Yerkovich et al.,1997).

Increased levels of IL-6 were observed in the spleens andlungs of clinically infected mice; however, no significant lev-els of IL-6 transcripts were detected in sub-clinically infectedmice. The function of IL-6 as observed in this study is notwell defined. Clearly, IL-6 is produced in response to clini-cal MCMV infection, especially in the spleen. However, thelack of detectable IL-6 production during sub-clinical infec-tion is noteworthy, and suggests the possibility that IL-6 isupregulated only in response to more severe infection. Fur-ther experiments should be conducted to better understandthe role of IL-6 in MCMV infection.

pro-d ands eens ud-i ofI sei air-i + Tc IL-1 iti ryc ies,t ini-c n ofT gsa ingp heseo

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. Discussion

The goal of this study was to develop a quantitaeal-time PCR assay to investigate organ-specific cytoesponse during both clinical and sub-clinical MCMV infion, and to determine the relationship between cytoranscription and viral load and disease severity. Prevtudies on the role of cytokines during MCMV infectiave looked at the systemic cytokine response in the sf infected mice using ELISA based assays (Pomeroy et al998; Wu et al., 2001), or at tissue levels (spleen and lungytokines using RNA protection assays or reverse transion of RNA by conventional PCR (Karupiah et al., 1998; Wt al., 2001). Real-time PCR (Heid et al., 1996; Kipar et a001) allowed the analysis of cytokine transcription and v

iters in specific organs in a time dependant manner.The data presented here indicate that clinical MC

nfection induces a pro-inflammatory response in the lupleens and livers characterized by the production of IN�,NF-� and IL-6 at 5 days post-infection. Studies on se

evels of INF� have found levels to peak 2 days after infec

An anti-inflammatory response characterized by theuction of IL-10 was also observed in the lungs, liverspleens of clinically infected mice. Several roles have buggested for IL-10 during MCMV infection. Previous stes (Redpath et al., 1999) have reported that productionL-10 early during MCMV infection results in a decrean expression of MHC class II proteins, therefore impng the ability of macrophages to present antigen to CD4ells and diminishing antiviral response. Production of0 later during MCMV infection may also serve to lim

nflammation by inhibiting the production of inflammatoytokines, and may help limit host damage. In our studhe elevated transcription of IL-10 in the organs of clally infected mice paralleled the elevated transcriptioNF-� (IL-10 and TNF-� levels peaked at day 5 in lunnd livers) suggesting a possible role for IL-10 in limitro-inflammatory cytokine-induced tissue damage in trgans.

Our findings indicate that IL-2 does not play a major rn the host response to MCMV infection. These findire supported by others who found depressed levels o

128 Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122–129

2 production in vitro in cell cultures and serum (Blackett andMims, 1988; Karupiah et al., 1998).

Using qRT-PCR, MCMV DNA was detected in the lungs,livers and spleens of sub-clinically infected animals evenin the absence of clinical signs. In most organs, inductionof cytokine transcription during sub-clinical infection was2–100-fold lower than during clinical infection, with TNF�,INF� and IL-10 being the major cytokines produced. Mostof the cytokine response to sub-clinical MCMV infectionoccurred in the spleens at day 5, and in the salivary glandslater, at day 15 post-infection.

The peaks in TNF-� and INF-� production found in allorgans correlated with the timing of increases in viral load inthese organs and with the severity of the signs observed (clini-cal versus sub-clinical infection). MCMV DNA was detectedin all organs analyzed during clinical infection, and viral titersclosely correlated with the level of inflammatory cytokines.The strongest associations between viral load and cytokinetranscription were found in the livers, spleens and salivaryglands (p ≤ 0.005). These correlations were observed in eachof the mice in this study. Despite these associations, a substan-tial variation in cytokine response and viral load was observedamong the different individual mice. This individual variationis clearly reflected in the deviation from the overall mean val-ues suggesting that induction of cytokines in specific organsis complex and may be unique to each specific animal.

nts as f theo micl thiss onseo itet wasc loadi

an-s ingq ccu-r wsu n andt imep tiver rip-t iteo MVD thes thod-o hec V ist ilityo lP uiresm RT-P

anda ine

response to MCMV. By using this approach, these studieshave shown that the induction pattern of cytokines is organ-specific and that cytokine production correlates with organ-specific viral load and disease severity. Additional studiesare in progress in our laboratory to further define the kinet-ics and nature of organ-specific cytokine response in MCMVinfection and to correlate organ-specific cytokine transcrip-tion with organ-specific cytokine protein levels.

Acknowledgements

The authors thank Ms. Tammi Olineka and Mr. MarkoEstrada (Lucy Whittier Molecular Core Facility, School ofVeterinary Medicine at UC Davis) for excellent technicalassistance with RNA extraction and cDNA synthesis. We aregrateful to Dr. Christian Leutenegger (Lucy Whittier Molec-ular Core Facility) for valuable suggestions and advise onthe TaqMan experiments. We thank Dr. Laurel Beckett forexcellent assistance with the statistical analysis.

This work was supported in part by a Merit Grant (to CP)from the Department of Veteran Affairs.

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