Journal of Virological Methods 131 (2006) 122129

Use of quantitative real-time PCR (qRT-PCR) to measure cytokine transcription and viral load in murine cytomegalovirus infection
Yajarayma J. Tang-Feldman a,b, , Angela Wojtowicz a,b , G. Raymond Lochhead a,b , Merica A. Hale a,b , Yueju Li c , Claire Pomeroy a,b,d
b

Department of Internal Medicine, Division of Infectious and Immunologic Diseases, University of California, Davis Health System, Sacramento, CA, USA Department of Medical Microbiology and Immunology, University of California, Davis, Davis, Sacramento, CA, USA c Department of Public Health, School of Medicine, University of California, Davis, Davis, CA, USA d 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 2005 Available online 2 September 2005

Abstract A quantitative real-time PCR (qRT-PCR) assay was developed to measure cytokine transcription proles and viral load during sub-clinical and clinical infection with murine cytomegalovirus (MCMV). Primers/uorogenic probes specic for mouse cytokines and for the immediate early gene 1 (IE1) of MCMV were used to quantitate cytokine responses and viral load in various organs of MCMV infected mice. Increased mRNA levels of TNF- , INF- and IL-10 were detected in the spleens, lungs and livers of clinically infected mice at 5 days post-infection. Transcription of these cytokines was 25-fold lower (p = 0.07 for each cytokine) in the spleens and 10100-fold lower in the lungs (p = 0.03 for INF , not signicant for IL-10 and TNF ) and livers (p < 0.05 for each cytokine) of sub-clinically infected mice. Clinical MCMV infection induced high levels of IL-6 in the lungs and spleens of infected animals, while no signicant transcription of IL-6 was detected in any organ during sub-clinical infection (p < 0.05). The timing of peak amounts of INF- , IL-10 and IL-6 observed in the spleens of clinically infected mice correlated with high viral loads in these organs. Cytokine expression rose in the salivary glands later, at day 15, corresponding to the increase in salivary gland viral load. The qRT-PCR demonstrates that infection with MCMV induces an organ-specic cytokine response characterized by the production of TNF- , INF- , IL-6 and IL-10 which correlates with severity of the disease (sub-clinical versus clinical) and with viral load. In summary, qRT-PCR is a sensitive and accurate method to study MCMV infection and host responses to the virus. 2005 Elsevier B.V. All rights reserved.
Keywords: MCMV; Quantitative RT-PCR; Cytokine response; Viral load

1. Introduction Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in immunocompromised patients, especially those with HIV/AIDS infection and in bone marThis work was presented in part at the 42nd Annual Meeting of the Infectious Diseases Society of America, Boston, MA, USA, 2004. Corresponding author. Present address: University of California, Davis Health System, 4150 V Street, Suite 1100, Sacramento, CA, USA. Tel.: +1 916 734 3578; fax: +1 916 734 7055. E-mail addresses: yjtangfeldman@ucdavis.edu (Y.J. Tang-Feldman), claire.pomeroy@ucdmc.ucdavis.edu (C. Pomeroy). 0166-0934/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.07.013

row and solid organ transplant recipients (Ho, 1995). CMV is also the most common infectious cause of congenital malformations (Stagno et al., 1986). CMV infection is most commonly acquired during childhood, as reected by seropositivity prevalence rates of 4095% in adults around the world. Indeed, most CMV infections are sub-clinical and asymptomatic, and therefore go unrecognized. Murine (mouse) models of CMV (MCMV) infection are useful for studying host response to the virus because many of the immune responses parallel those of humans. The murine model is an excellent tool to study the course of both clinical and sub-clinical infection, as well as viral latency and reac-

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tivation. Research on MCMV has provided signicant information on the pathogenesis of clinical CMV disease, including the role played by cytokines in host defense (Orange and Biron, 1996a; Pavic et al., 1993; Pomeroy et al., 1991). Cytokines are critical in the defense against MCMV. TNFhas been reported to limit titers of MCMV in organs in vivo, and has been associated with protective effects during acute infection in adult BALB/c mice (Orange and Biron, 1996b; Pavic et al., 1993). However, some studies have shown that neutralization of TNF- in MCMV infected mice had no effect on viral replication or disease outcome (Shanley et al., 1994). INF- has been associated with antiviral activities during MCMV infection in both BALB/c and C57BL/6 mice (Orange and Biron, 1996b). Administration of high doses of INF- increased morbidity and mortality in BALB/c mice suggesting that the cytokine has both benecial and detrimental effects on the host (Pomeroy et al., 1998). IL-6, IL-4 and IL-10 have also been reported to play a role in MCMV infection (Geist and Hinde, 2001). Studies of cytokine production in response to MCMV infection have usually assessed serum levels or systemic cytokine response in clinical, symptomatic infections using ELISA based assays. Others have used RNA protection assays or reverse transcriptase PCR to study cytokine expression in bronchoalveolar lavage and spleen (Karupiah et al., 1998; Wu et al., 2001). Little is known about organ-specic cytokine response during MCMV infection, primarily due to limitations of the techniques available. In the past 10 years, quantitative real-time PCR (qRTPCR) has become one of the techniques of choice to study gene expression in cells and tissues. The use of uorescent labeled probes increases the sensitivity and specicity of the system allowing detection of very minute amounts of transcripts. This method has been used to study cytokine response to M. bovis in guinea pigs (Kawahara et al., 2002), cytokine and chemokine transcription in response to H. inuenzae in humans (Tong et al., 2001), and to assess cytokine mRNA levels in feline monocytes (Kipar et al., 2001). qRT-PCR assays have also been used to quantitate viral loads in a number of viral infections including CMV (Wheat et al., 2003; Vliegen et al., 2003; Gault et al., 2001; Jebbink et al., 2003). In this study, a quantitative real-time PCR assay was developed to analyze the organ-specic transcription levels of a panel of cytokines (IL-2, IL-6, IL-10, TNF- and INF- ) in selected organs during sub-clinical/asymptomatic infection and clinical infection with MCMV. These cytokine transcription patterns were then correlated with the organ-specic viral load.

All mice were housed in AAALAC-approved SPF facilities at our institution and had free access to food and water. Animal studies and protocols were approved by the UC Davis Institutional Animal Care and Use Committee (IACUC). 2.2. Virus and infection MCMV Smith strain was obtained from American Type Culture Collection (ATCC) (Manassas, VA). The virus was maintained by salivary gland passage in BALB/c mice, and a salivary gland homogenate (SGV) was prepared in RPMI (Gibco Laboratories, Grand Island, NY) as described elsewhere (Pomeroy et al., 1991). To determine the sub-lethal dose to establish a clinical infection, a LD50 of the SGV was performed using standard protocols (Pomeroy et al., 1991). Mice were infected by intraperitoneal (i.p.) injection of 0.2 ml of a sub-lethal dose (a dose of virus appropriately adjusted to cause severe disease but not lethal infection, 104 PFU) of the SGV in RPMI. This dose was 2-fold lower than the LD50 dose. Clinically infected mice were observed and scored daily for signs: lethargy, weight loss, rufed fur, hunched posture and squinty eyes. Groups of mice (n = 8) were sacriced at days 5 and 15 post-infection for analysis of cytokine expression and viral load. To establish a sub-clinical infection, mice received by i.p. route 0.2 ml of a sub-clinical dose of the SGV (300 PFU), an amount previously found to infect mice without causing any clinical signs (unpublished data). Sub-clinically infected mice were sacriced at days 5 and 15 post-infection. In each experiment, a control group (uninfected mice) received 0.2 ml of RPMI intraperitoneally, and had their organs removed and analyzed for cytokine expression, as in the infected groups. Signs of MCMV infection usually appear 2 days after clinical infection and continue for about 5 more days with recovery of the mice starting around days 810 post-infection. The time points used in this study encompass the peak of acute signs (5 days) to the complete resolution of clinically apparent signs (15 days). 2.3. Tissue collection At each time point, a group of mice (n = 8) was sacriced and samples of lungs, livers, spleens, salivary glands and small intestines removed and placed in Nucleic Acid Purication Lysis Solution (Applied Biosystems, Foster City, CA) for DNA and RNA extraction. 2.4. Nucleic acid extraction and cDNA synthesis DNA was extracted from each organ with the DNeasy Tissue Kit (Qiagen, Valencia, CA) following the manufacturers instructions. RNA extraction and cDNA synthesis were performed at the Molecular Core Facility, School of Veterinary Medicine at our institution using the 6700 RNA extraction apparatus (Applied Biosystems) as per instructions and guidelines. Briey, cDNA was synthesized using 100 units

2. Materials and methods 2.1. Mice Pathogen-free 34 weeks old BALB/c female mice were purchased from Harlan Sprague Dawley (Indianapolis, IN).

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of SuperScript II (Invitrogen, Carlsbad, CA), random hexamer primers (300 ng), 10 U of RNaseOut (Invitrogen), 1 mM dNTPs and 20 l of the RNA preparation in a nal volume of 40 l. After 50 min of incubation at 42 C, 10 l of water were added and the reaction was terminated by heating for 5 min at 95 C. 2.5. Real-time PCR of cytokines Quantitative transcription prole of IL-2, IL-6, IL-10, TNF- and INF- in each organ was determined by qRTPCR using the Assay on Demand kits (Applied Biosystems) with the respective primers/uorogenic probe mix specic for each mouse cytokine. In addition, transcription of the housekeeping genes, GAPDH and HPRT, in each organ was determined using specic primer/uorogenic probe mix (Applied Biosystems). Real-time PCR reactions were set up in duplicates for each of the cytokines and housekeeping genes in each organ analyzed. Amplication conditions were identical for all reactions and consisted of: 2 min at 50 C, 10 min at 95 C, 40 cycles of 15 s at 95 C and 60 s at 60 C. Reaction samples had a nal volume of 6 l consisting of 3.5 l of Universal Master mix containing the specic primer/probe mix (Applied Biosystems) and 2.5 l of the respective cDNA. Amplications were run in an ABI Prism 7900 Sequence Detection System (Applied Biosystems). Final quantication was performed as described elsewhere using the comparative CT method (Kipar et al., 2001). For each experimental sample, the difference between the target CT value and the CT value of the most stable housekeeping gene for each tissue type was used to normalize for differences in the amount of total nucleic acid added to each reaction and the efciency of the RT step ( CT ). For relative quantication by the comparative CT method, values are then expressed relative to a reference sample called the calibrator. The calibrator is the weakest signal from the normalization ( CT ) in each tissue type. The CT for each experimental sample was subtracted from the CT of the calibrator ( CT ). The amount of target (linear value) normalized to an internal control or housekeeping gene and relative to the calibrator was determined by 2 CT . The linear expression of each gene in each tissue was then calculated by subtracting the linear expression value of each gene in uninfected mice (baseline value) from that of the infected mice. 2.6. Amplication and cloning of MCMV immediate early 1 (IE1) gene DNA was extracted from MCMV Smith strain (ATCC) using the QIAmp DNA Blood Mini Kit (Qiagen) as per manufacturers instructions. Oligonucleotide primers used for amplication of the IE1 gene were: forward primer 5 -TCAGCCATCAACTCTGCTACCAAC-3 and reverse primer 5 ATCTGAAACAGCCGTATATCATCTTG-3 . Amplication was performed with the HotStarTaq Master Mix kit (Qiagen)

as per instructions. Samples were set up in 50 l nal volumes containing 25 l of the master mix, 30 pmoles of the respective forward and reverse primer, and the reaction was made up to 50 l with sterile water. PCR conditions included a hot start step at 95 C for 10 min, followed by 40 cycles consisting of 95 C for 45 s, 55 C for 45 s and 72 C for 1 min with a nal extension at 72 C for 7 min. The respective amplicon was cloned into a pCR II TOPO vector using the TOPO TA Cloning kit (Invitrogen, USA) as per manufacturers instructions. Plasmids were puried using the Qiagen Plasmid Mini Prep (Qiagen) and quantication was done by spectrophotometry. 2.7. Quantication of MCMV using real-time PCR Serial 10-fold dilutions of the plasmid containing the IE1 insert 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 from 2 to 2 109 copies. The forward and reverse primers listed above and the TaqMan probe were designed using the Primer Express Software (Applied Biosystems). The sequence of the TaqMan probe was 5 -TTCTCTGTCAGCTAGCCAATGATATCTTCGAGC-3 . For real-time PCR, the probe was labeled at the 5 end with the reporter dye FAM and at the 3 end with the quencher dye TAMRA. Real-time PCR was performed using the TaqMan Universal Master Mix (Applied Biosystems). Reactions were set up in triplicates in 12 l volumes consisting of 7 l of the cocktail containing the Universal Master mix, 400 nM of each primer, 80 nM of the TaqMan probe and 5 l of the respective plasmid dilution. Amplication conditions consisted of 2 min at 50 C, 10 min at 95 C, and 40 cycles of 15 s at 95 C and 60 s at 60 C. A standard curve was constructed by plotting average CT values against the logarithm of the target template molecules obtained from the plasmid, followed by a sum of least squares regression analysis (Fig. 1). Target copy numbers in tissue samples were calculated using the equations obtained in regression analysis. Results were expressed as

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

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DNA copies/100 mg of tissue (Vliegen et al., 2003). Since DNA yield per 100 mg of tissue differs from tissue to tissue, DNA was extracted from 100 mg of each tissue and analyzed using the Qiagen DNeasy kit (Qiagen). All extractions were done in quadruplicate and the average was used to determine the DNA yield/100 mg of each tissue type. Because only 1 g of DNA is used in qRT-PCR, the number of MCMV copies/100 mg of tissue was considered to equal the number of copies obtained in the real-time PCR reaction multiplied by the amount of total DNA obtained for each specic tissue type. 2.8. Statistical analysis To determine if the cytokine response during clinical infection was signicantly greater than during sub-clinical infection, two-sample t-tests were used on double-log transformed data, separately for each cytokine in each organ; 95% condence intervals for the difference in means were calculated on the double-log transformed scale and then transformed back to give condence intervals in the original units of measurements. Similarly, cytokine response was compared for each pair of time points using t-tests and 95% condence intervals on the double-log scale. Pearson correlations were calculated between cytokine and viral load. All data were plotted to check normality and non-parametric alternative procedures (Wilcoxon rank sum test, Spearman rank correlation) were carried out if the data suggested possible violations of distribution assumptions even after log or double-log transformation. All tests were two-sided at level 0.05, and all analyses were performed using SAS Version 8.2 (SAS Institute Inc., 19992001).

Table 1 Clinical signs of MCMV infection in BALB/c mice Day post-infection 1 Weight lossa (%) 2.5 +1 +1 +1 0 2 10.1 +1 +1 +1 +1 3 13.4 +2 +2 +2 +2 5 23.2 +3 +3 +3 +2 8 21.5 +2 +2 +1 0 15 5.0 0 0 0 0

Signsb Lethargic Rufed fur Hunched posture Squinty eyes

Score: 0, no signs observed; +1, slightly lethargic, rufed primarily around neck, hunched while sitting; +2, lethargic, rufed fur around neck area and torso, back bone prominent when sitting, squinty eyes; +3, very lethargic, rufed 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 remaining in all groups at the respective time points. b Signs and severity represent compiled observations of the mice remaining in all groups at the respective time points.

3.2. Quantication of cytokine transcription during sub-clinical and clinical infection measured by qRT-PCR Cytokine response in the various organs was assessed at 5 and 15 days post-infection. Linear expression values of cytokines in infected mice were standardized to the background or baseline expression in uninfected mice. A substantial variability in cytokine response in each organ was observed in individual animals. TNF- , INF- and IL-6 transcripts reached highest levels in the lungs and livers of clinically infected mice at days 5 post-infection (Table 2). In the spleens, levels of TNF- and IL-6 rose at day 5 postinfection and remained elevated throughout the study period. Transcription of these cytokines at day 5 post-infection in sub-clinically infected mice was 2100-fold lower than in clinical infection. IL-6 transcription levels were also elevated in the spleens and lungs of clinically infected mice, while no signicant transcription of IL-6 was detected in any organs of the sub-clinically infected mice (Table 2). In contrast to other organs, cytokine expression levels in the salivary glands of sub-clinically infected mice did not increase signicantly at day 5 post-infection. Expression of TNF- , INF- and IL-10 in salivary glands of clinically infected mice was detected at day 5, remaining signicantly elevated above baseline at day 15 (p < 0.04) (Table 2). Levels of these cytokines in salivary glands of clinically infected mice were 5200-fold higher than in sub-clinically infected mice at both day 5 and day 15 (Table 2). The severity of signs observed in clinically infected mice correlated with peak TNF- and INF- responses (day 5) and highest viral loads in the lungs and livers, and peak INF- response in the spleens (day 5). No cytokine mRNA transcripts were detected in the small intestines. IL-2 was not detected in any organ at the time points analyzed (data not shown).

3. Results 3.1. Establishment of sub-clinical and clinical infection with MCMV Little is known about cytokine response during asymptomatic MCMV infection. To determine the usefulness of the qRT-PCR assay to study cytokine responses during sub-clinical infection, two groups of mice were infected: clinically infected mice received a sub-lethal dose of the MCMV-SGV (104 PFU) capable of causing severe disease but no lethal infection; sub-clinically infected mice received a dose of the MCMV-SGV (300 PFU) previously found to cause infection but no apparent clinical signs (unpublished data). Clinically infected mice developed typical signs of the disease. Signs of MCMV infection appeared at day 1 post-infection, and peaked around day 5 after infection. By day 10 post-infection most clinically apparent signs subsided and by day 15 mice appeared clinically recovered (Table 1). No clinical manifestation of disease was observed in the sub-clinically infected mice.

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Table 2 TNF- , INF- , IL-6 and IL-10 transcription prole and viral load at 5 and 15 days post-sub-clinical and clinical infection with MCMV Tissue Day post-infection Linear valueb TNF- (S.D.)a Spleen Sub-clinical Clinical p Sub-clinical Clinical p Lung Sub-clinical Clinical p Sub-clinical Clinical p Liver Sub-clinical Clinical p Sub-clinical Clinical p Salivary gland Sub-clinical Clinical p Sub-clinical Clinical p 5 5 15 15 612 (1538) 2495 (2100) 0.07 217 (238) 3716 (5068) 0.02 69 (42) 3821 (5300) 0.007 6 (11) 508 (1200) 0.001 25 (32) 5770 (9374) 0.007 65 (59) 59 (176) 0.13 10 (76) 2131 (2068) 0.03 245 (186) 1128 (1590) 0.007 IL-6 (S.D.)a 1 (1) 395 (392) 0.003 2 (4) 2634 (3370) 0.0015 0 (3) 64 (108) 0.23 5 (6) 0 (3) 0.009 0 91 (160) 0.002 0 24 (11) 0.01 0 12 (15) 0.01 0 6 (8) 0.04 IL-10 (S.D.)a 99 (215) 571 (440) 0.08 109 (90) 88 (110) 0.91 196 (151) 832 (1450) 0.29 7 (8) 933 (1060) 0.38 64 (44) 924 (1800) 0.03 7 (2) 3 (15) 0.38 19 (14) 630 (1430) 0.099 25 (28) 703 (1013) 0.001 INF- (S.D.)a 1069 (2700) 1744 (2150) 0.16 206 (141) 411 (595) 0.98 27 (18) 350 (443) 0.03 1 (2) 102 (148) 0.15 6 (10) 600 (1080) 0.01 5 (2) 8 (21) 0.11 7 (7) 95 (192) 0.05 13 (7) 149 (252) 0.003 1.1 106 4.8 107 0.036 3.9 102 5.1 106 0.007 6.8 102 1.6 105 0.042 0 1.5 102 0.0001 1.5 103 7.5 106 0.012 0 7.4 103 0.001 1.1 102 1.8 105 0.005 1.6 105 1.0 107 0.08 + Viral loadc Signsd

5 5 15 15

5 5 15 15

5 5 15 15

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 (see Table 1); (), no symptoms observed.

3.3. Quantication of MCMV viral load in specic organs during sub-clinical and clinical infection In our experiments, none of the sub-clinically infected mice showed signs of MCMV disease. Despite this, MCMV DNA was detected in the lungs, livers and spleens at day 5 post-infection (Table 2). By day 15 post-infection, viral DNA had considerably decreased in these organs. In contrast, signicant viral DNA was detected in the salivary glands of subclinically infected mice at day 15 post-infection, although levels remained three logs below those found in the salivary glands of clinically infected animals (Table 2). Viral load was 1010,000-fold higher in most organs during clinical infection compared to sub-clinical infection. DNA copies of MCMV in the lungs, livers and spleens of clinically infected mice were highest at day 5 post-infection and ranged from 105 to 107 DNA copies/100 mg of tissue (Table 2). By day 15, viral DNA had decreased by 1000-fold in lungs and livers of clinically infected mice, while viral load

in the spleens remained relatively high throughout the study period in parallel with high levels of cytokines. In contrast, in the salivary glands, a higher viral load was detected later, at day 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) 122129 Table 3 Organ-specic correlations between viral load and cytokine response (Spearman rank correlation, signicance, p = 0.05 or lower; trends, p < 0.10) Organ Liver Lung Salivary gland Small intestine Spleen IL-10 (p-value) 0.52 (<0.001) 0.28 (0.066) 0.53 (<0.001) 0.20 0.41 (0.004) IL-6 (p-value) 0.54 (<0.001) 0.15 0.53 (<0.001) 0.05 0.42 (0.003) INF- (p-value) 0.47 (0.001) 0.20 0.62 (<0.001) 0.12 0.54 (<0.001)

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TNF- (p-value) 0.49 (<0.001) 0.43 (0.005) 0.52 (<0.001) 0.21 0.45 (0.002)

detected in the salivary glands of sub-clinically infected mice at 15 days after infection (Table 2). 3.4. Correlation of organ-specic cytokine transcription and viral load Signicant correlation was found between extent of cytokine production and viral load in spleens (p < 0.005 for IL-6, IL-10, INF- and TNF ), livers (p < 0.005 for IL-6, IL-10, INF- and TNF- ), lungs (p = 0.005 for TNF- ) and salivary glands (p < 0.001 for IL-6, IL-10, INF- and TNF- ) (Table 3). Notably, cytokine levels peaked in lungs, and livers at day 5 when viral loads were higher, and persisted elevated longer in the spleens, where viral load remained elevated longer. In contrast, cytokine levels (IL-10 and INF- ) stayed low in salivary glands early when viral replication was limited, and rose later at day 15, in concert with the later increases in viral load. Viral DNA copy numbers in small intestines were 100010,000-fold lower than in the spleens, livers and lungs. The levels of cytokine transcription in these organs was 10100-fold lower than in spleens, livers and lungs (data not shown). These ndings are consistent with organ-specic cytokine responses that parallel organ-specic differences in extent and timing of viral replication.

4. Discussion The goal of this study was to develop a quantitative real-time PCR assay to investigate organ-specic cytokine response during both clinical and sub-clinical MCMV infection, and to determine the relationship between cytokine transcription and viral load and disease severity. Previous studies on the role of cytokines during MCMV infection have looked at the systemic cytokine response in the serum of infected mice using ELISA based assays (Pomeroy et al., 1998; Wu et al., 2001), or at tissue levels (spleen and lung) of cytokines using RNA protection assays or reverse transcription of RNA by conventional PCR (Karupiah et al., 1998; Wu et al., 2001). Real-time PCR (Heid et al., 1996; Kipar et al., 2001) allowed the analysis of cytokine transcription and viral titers in specic organs in a time dependant manner. The data presented here indicate that clinical MCMV infection induces a pro-inammatory response in the lungs, spleens and livers characterized by the production of INF- , TNF- and IL-6 at 5 days post-infection. Studies on serum levels of INF have found levels to peak 2 days after infection

in BALB/c mice, probably due to an initial response mediated by natural killer (NK) cells (Orange et al., 1995; Shanley et al., 1994). High levels of INF- have also been detected in the serum of infected mice up to 67 days after infection (Pomeroy et al., 1998). Elevated levels of TNF- were detected at day 5 during clinical infection in all the organs analyzed. Transcription of TNF- was markedly increased in the spleens, lungs and livers during early stages of infection (day 5) and remained elevated in the spleens throughout the study period in parallel with a high viral load. These results are consistent with previous reports of high levels of TNF- in the serum of acutely infected mice, suggesting a role for TNF- in the initial control of the infection (Trgovcich et al., 2000; Yerkovich et al., 1997). Increased levels of IL-6 were observed in the spleens and lungs of clinically infected mice; however, no signicant levels of IL-6 transcripts were detected in sub-clinically infected mice. The function of IL-6 as observed in this study is not well dened. Clearly, IL-6 is produced in response to clinical MCMV infection, especially in the spleen. However, the lack of detectable IL-6 production during sub-clinical infection is noteworthy, and suggests the possibility that IL-6 is upregulated only in response to more severe infection. Further experiments should be conducted to better understand the role of IL-6 in MCMV infection. An anti-inammatory response characterized by the production of IL-10 was also observed in the lungs, livers and spleens of clinically infected mice. Several roles have been suggested for IL-10 during MCMV infection. Previous studies (Redpath et al., 1999) have reported that production of IL-10 early during MCMV infection results in a decrease in expression of MHC class II proteins, therefore impairing the ability of macrophages to present antigen to CD4 + T cells and diminishing antiviral response. Production of IL10 later during MCMV infection may also serve to limit inammation by inhibiting the production of inammatory cytokines, and may help limit host damage. In our studies, the elevated transcription of IL-10 in the organs of clinically infected mice paralleled the elevated transcription of TNF- (IL-10 and TNF- levels peaked at day 5 in lungs and livers) suggesting a possible role for IL-10 in limiting pro-inammatory cytokine-induced tissue damage in these organs. Our ndings indicate that IL-2 does not play a major role in the host response to MCMV infection. These ndings are supported by others who found depressed levels of IL-

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2 production in vitro in cell cultures and serum (Blackett and Mims, 1988; Karupiah et al., 1998). Using qRT-PCR, MCMV DNA was detected in the lungs, livers and spleens of sub-clinically infected animals even in the absence of clinical signs. In most organs, induction of cytokine transcription during sub-clinical infection was 2100-fold lower than during clinical infection, with TNF , INF and IL-10 being the major cytokines produced. Most of the cytokine response to sub-clinical MCMV infection occurred in the spleens at day 5, and in the salivary glands later, at day 15 post-infection. The peaks in TNF- and INF- production found in all organs correlated with the timing of increases in viral load in these organs and with the severity of the signs observed (clinical versus sub-clinical infection). MCMV DNA was detected in all organs analyzed during clinical infection, and viral titers closely correlated with the level of inammatory cytokines. The strongest associations between viral load and cytokine transcription were found in the livers, spleens and salivary glands (p 0.005). These correlations were observed in each of the mice in this study. Despite these associations, a substantial variation in cytokine response and viral load was observed among the different individual mice. This individual variation is clearly reected in the deviation from the overall mean values suggesting that induction of cytokines in specic organs is complex and may be unique to each specic animal. Measurement of cytokines levels in serum represents a systemic response to the infection and it is a reection of the overall cytokine production in the organism. These systemic levels may show less variability; however, the goal of this study was to develop a method to investigate the response of specic organs to MCMV infection. Importantly, despite these variations we found that in individual mice, there was close correlation between cytokine production and viral load in individual organs in individual mice. To our knowledge, this is the rst study to analyze organspecic cytokine production in parallel with viral load using quantitative real-time PCR. This method offers a more accurate depiction of cytokine prole during infection as it allows us to specically measure the response in each organ and to correlate it with the organ-specic viral load at each time point. Furthermore, the increased sensitivity of quantitative real-time PCR allowed the detection of low-level transcription of cytokines in sub-clinically infected animals in spite of the absence of clinical signs. The detection of MCMV DNA (as low as 150 DNA copies/100 mg of tissue in the small intestines) demonstrates the usefulness of this methodology in studying sub-clinical asymptomatic infections. The conventional plaque assay method to quantitate MCMV is time consuming and lacks the sensitivity and reproducibility of qRT-PCR (Vliegen et al., 2003). Although conventional PCR has been used to detect latent viral infection, it requires multiple steps, and it lacks the quantitative power of qRTPCR. In summary, quantitative real-time PCR is a sensitive and accurate method to study MCMV infection and cytokine

response to MCMV. By using this approach, these studies have shown that the induction pattern of cytokines is organspecic and that cytokine production correlates with organspecic viral load and disease severity. Additional studies are in progress in our laboratory to further dene the kinetics and nature of organ-specic cytokine response in MCMV infection and to correlate organ-specic cytokine transcription with organ-specic cytokine protein levels. Acknowledgements The authors thank Ms. Tammi Olineka and Mr. Marko Estrada (Lucy Whittier Molecular Core Facility, School of Veterinary Medicine at UC Davis) for excellent technical assistance with RNA extraction and cDNA synthesis. We are grateful to Dr. Christian Leutenegger (Lucy Whittier Molecular Core Facility) for valuable suggestions and advise on the TaqMan experiments. We thank Dr. Laurel Beckett for excellent assistance with the statistical analysis. This work was supported in part by a Merit Grant (to CP) from the Department of Veteran Affairs. References
Blackett, S., Mims, C.A., 1988. Studies of depressed interleukin-2 production by spleen cells from mice following infection with cytomegalovirus. Arch. Virol. 99, 1. Geist, L.J., Hinde, S.L., 2001. Susceptibility to cytomegalovirus infection may be dependent on the cytokine response to the virus. J. Invest. Med. 49, 434. Gault, E., Michel, Y., Dehee, A., Belabani, C., Nicolas, J.C., GarbargChenon, A., 2001. Quantication of human cytomegalovirus DNA by real-time PCR. J. Clin. Microbiol. 39, 772. Heid, C.A., Stevens, J., Livak, K.J., Williams, P.M., 1996. Real-time quantitative PCR. Genome Res. 6, 686. Ho, M., 1995. Cytomegalovirus. In: Mandell, G.L., Bennett, J.E., Dolin, R. (Eds.), Principles and Practice of Infectious Diseases, fourth ed. Churchill Livingstone, New York, p. 1351. Jebbink, J., Bai, X., Rogers, B., Dawson, D.B., Scheuermann, R.H., Domiati-Saad, R., 2003. Development of real-time PCR assay for the quantitative detection of Epstein-Barr Virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons. J. Mol. Diagn. 5, 15. Karupiah, G., Sacks, T.E., Klinman, D.M., Fredrickson, T.N., Hartley, J.W., Chen, J.H., Moser III, H.C., 1998. Murine cytomegalovirus infection-induced polyclonal B cell activation is independent of CD4 + T cells and CD40. Virology 240, 12. Kawahara, M., Nakasone, T., Honda, M., 2002. Dynamics of gamma interferon, interleukin-12 (IL-12), IL-10, and transforming growth factor mRNA expression in primary Mycobacterium bovis infection in guinea pigs measured by a real-time PCR uorogenic reverse transcriptionPCR Assay. Infect. Immun. 70, 6614. Kipar, A., Leutenegger, C.M., Hetzel, U., Akens, M.K., Mislin, C.N., Reinacher, M., Lutz, H., 2001. Cytokine mRNA levels in isolated feline monocytes. Vet. Immunol. Immunopathol. 78, 305. Orange, J.S., Biron, C.A., 1996a. Characterization of early IL-12, INF , and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J. Immunol. 156, 4746. Orange, J.S., Biron, C.A., 1996b. An absolute and restricted requirement for IL-12 in natural killer cell INF- production and antiviral defense. J. Immunol. 156, 1138.

Y.J. Tang-Feldman et al. / Journal of Virological Methods 131 (2006) 122129 Orange, J.S., Wang, B., Terhorst, C., Biron, C.A., 1995. Requirement for natural killer cell-produced INF in defense against murine cytomegalovirus infection and enhancement of this defense pathway by IL-12 administration. J. Exp. Med. 182, 1045. Pavic, I., Polic, B., Crknovic, I., Luvin, P., Jonjie, S., Koszinowski, U.H., 1993. Participation of endogenous tumor necrosis factor- in the host resistance to cytomegalovirus infection. J. Gen. Virol. 74, 2215. Pomeroy, C., Delong, D., Clabots, C., Riciputi, P., Filice, G.A., 1998. Role of interferon- in murine cytomegalovirus infection. J. Lab. Clin. Med. 132, 124. Pomeroy, C., Hilleren, P.J., Jordan, M.C., 1991. Latent murine cytomegalovirus DNA in splenic stromal cells of mice. J. Virol. 65, 3330. Redpath, S., Angulo, A., Gascoigne, N.R., Ghazal, P., 1999. Murine cytomegalovirus infection down-regulates MHC Class II expression on macrophages by induction of IL-10. J. Immunol. 162, 6701. SAS Institute Inc., 19992001. SAS Version 8.2. SAS Institute Inc., Cary, NC. Shanley, J.D., Goff, E., Debs, R.J., Forman, S.J., 1994. The role of tumor necrosis factor- in acute murine cytomegalovirus infection in BALB/c mice. J. Infect. Dis. 169, 1088. Stagno, S., Pass, R.F., Cloud, G., Britt, W.J., Henderson, R.E., et al., 1986. Primary cytomegalovirus infection in pregnancy: incidence, transmission to fetus and clinical outcome. JAMA 256, 1904.

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Tong, H.H., Chen, Y., James, M., Van Deusen, J., Welling, D.B., DeMaria, T.F., 2001. Expression of cytokine and chemokine genes by human middle ear epithelial cells induced by formalin killed Haemophilus inuenzae or its lipooligosaccharide htrB and rfaD mutants. Infect. Immun. 69, 3678. Trgovcich, J., Stimac, D., Polic, B., Krmpotic, A., Pernjak-Pugel, E., Tomac, J., Hasan, M., Wraber, B., Jonjic, S., 2000. Immune responses and cytokine induction in the development of severe hepatitis during acute infection with murine cytomegalovirus. Arch. Virol. 145, 2601. Vliegen, I., Herngreen, S., Grauls, G., Bruggeman, C., Stassen, F., 2003. Improved detection and quantication of mouse cytomegalovirus by real-time PCR. Virus Res. 98, 17. Wheat, R.L., Clark, P.Y., Brown, M.G., 2003. Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR. J. Virol. Methods 112, 107. Wu, A.C., Puddington, L., Whiteley, H.E., Yimouyiannies, C.A., Scharamm, C.M., Mohammadu, F., Thrall, R.S., 2001. Murine cytometalovirus infections alters Th1/Th2 cytokine expression, decreases airway eosinophilia and enhanaces mucus production in allergic airway disease. J. Immunol. 167, 2798. Yerkovich, S.T., Olver, S.D., Lenzo, J.C., Peacock, C.D., Price, P., 1997. The roles of tumor necrosis factor- , interleukin-1 and interleukin-2 in murine cytomegalovirus infection. Immunolology 91, 45.