Microbes and Infection 8 (2006) 1330e1338 www.elsevier.com/locate/micinf Original article Non-invasive imaging for monitoring herpes simplex virus type 1 hematogenous infection

Javier S. Burgos a,*,1, Fernando Guzman-Sanchez a,1, Isabel Sastre a, Cristina Fillat b, Fernando Valdivieso a,*

a Departamento de Biologı´a Molecular and Centro de Biologı´a Molecular Severo Ochoa (C.S.I.C.eU.A.M.), Universidad Auto´noma de Madrid, Spain b Programa Gens i Malaltia, Centre de Regulacio´ Geno`mica-CRG-UPF, Barcelona, Spain

Received 16 November 2005; accepted 26 December 2005 Available online 24 March 2006

Abstract

Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the repli- cation and tropism of KOS strain HSV-1 viruses expressing the firefly reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection. Ó 2006 Elsevier SAS. All rights reserved.

Keywords: HSV-1; Bioluminescence imaging; Hematogenous infection; Firefly luciferase

1. Introduction enabled in vivo imaging of luciferase expression in living mice via the use of cooled charge-coupled device (CCD) cam- In vivo bioluminescence imaging is a high-throughput, sen- eras [1,2]. Firefly (Photinus pyralis) luciferase (FL), the sub- sitive imaging method potentially ideal for evaluating many strate of which is D-luciferin, has been used in several biological phenomena. Advances in biotechnology have imaging studies of this type [3,4]. This enzyme has minimal background activity, can cross the cell membrane and can even penetrate into the bloodebrain barrier after intraperito- Abbreviations: CCD, cooled charge-coupled device ; FL, firefly neal (i.p.) or intravenous (i.v.) injection into mice. It can there- luciferase; HSV-1, herpes simplex virus type 1; i.p., intraperitoneal; PFU, fore be imaged in any organ [5]. Establishing the optimal plaque forming units; ROI, region-of-interest. conditions and potential limitations of this novel technique * Corresponding authors. Lab. CX340, Centro de Biologı´a Molecular, may allow its use in the evaluation of therapeutic responses Universidad Auto´noma de Madrid, 28049 Cantoblanco, Madrid, Spain. in preclinical studies. Tel.: þ34 914978471; fax: þ34 914974870. E-mail addresses: [email protected] (J.S. Burgos), fvaldivieso@cbm. Studies of pathogens in small animal models (generally uam.es (F. Valdivieso). mice) usually depend on the observation of clinical symptoms, 1 Both the authors contributed equally to this work. the sacrifice of the experimental animals, and the harvesting of

1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.12.021 J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338 1331 organs and tissues for histopathological examination or their from KOS/Dlux/oriL HSV-1-infected mice was also evaluated. use in molecular assays. The major drawback is that sequential Correlations were sought between the results provided by sacrifice precludes any subsequent observation of the microbi- these techniques. The results show that while bioluminescence ological, clinical, behavioral or other outcomes in the mice is promising, precise viral tracking and the exact quantification thus used. Animal-to-animal variations in hostepathogen in- of viral titers require combinations of these methodologies. teractions and therapeutic response are therefore commonly missed. The study of herpes simplex virus (HSV) infection 2. Materials and methods in living mice has been no exception. However, a powerful bioluminescence technique for monitoring this infection has 2.1. Recombinant virus recently been developed [6], and has in fact already been used to study HSV-1 pathogenesis by introducing firefly and HSV-1 KOS/Dlux/oriL (kindly supplied by Dr. D.A. Leib, Renilla luciferase genes into the viral genome [7]. Although Washington University, US) was constructed by introducing in this pioneering study the mice were infected via a number a cassette encoding the divergent UL29 and UL30 promoters of routes, the authors focused on ocular inoculation and the and the oriL region regulating FL into the viral genome at use of FL. Bioluminescence imaging of Renilla luciferase a site between UL49 and UL50 [6]. This virus strain was se- has been reported after systemic delivery of coelenterazine lected to be able to compare the results from this study with [8], but the above authors were unable to detect the activity already published data [12e14,18] and also because KOS of this reporter protein in the footpads or eyes of mice infected can be considered a prototypic laboratory strain of HSV-1. with KOS/Dlux/oriL HSV-1 [7]. The virus was propagated in confluent monolayers of Vero Interest on the hematogenous route of HSV infection has cells; titers were determined by plaque assays [12]. increased in recent years but it remains understudied e espe- cially with new imaging technologies. This type of infection 2.2. Inoculation and dissection has been widely reported in young [9e11] and adult animals [12e14] and is the main pathway by which HSV-1 infects ne- Experiments were performed in accordance with the guide- onates [15] and immunosuppressed humans [16]. The impor- lines of the European Community Animals Act (Scientific Pro- tance of this route resides in the fact that it is more effective cedures) of 1986. All the animals underwent a period of for the colonization of brain than alternative neural routes; quarantine. Strict precautions were taken to prevent contami- several host factors have already been found involved in hema- nation during inoculation and dissection. Nine-week-old out- togenous neuroinvasion [12e14]. Bioluminescence imaging bred CD-1 female mice (body weight, 26e28 g; Charles might be extremely useful when studying hematogenous River Laboratories, MA, US) (n ¼ 36) were i.p.-inoculated HSV-1 infection, since it potentially allows the non-invasive with 2 107 plaque forming units (PFU) of KOS/Dlux/oriL examination of intact organs and the recording of changes HSV-1 as previously described [12]. The mouse strain and over short periods of time. The observation and quantification the female gender were selected in order to compare the re- of in vivo light production rely on the spatial and temporal sults with already published data [7]. The female gender was distribution of photons emitted by the reporter in cells express- also used because this sex shows greater viral infectivity ing luciferase in the living animal. Bioluminescence imaging than males [14]. All the mice were marked, examined and an- could also be used to detect the spread of viruses to unexpected alyzed individually. Mock-infected animals were employed as anatomical sites; standard molecular techniques for detecting controls (used in parallel in all experiments). Mice were culled the virus require that organs be isolated whereas non-invasive at 0.8, 2, 3, 4, 5.7 and 7 days after inoculation. Organs (whole imaging technology allows whole-animal screening. blood, mesenteric lymph nodes, adrenal glands, ovaries, spinal In the present study, bioluminescence imaging was used to cord, trigeminal ganglia and brain) were dissected out and investigate changes in HSV-1 replication and tropism in hem- frozen. atogenously infected mice. A KOS/Dlux/oriL HSV-1 express- ing luciferase was used for this purpose; this reporter virus has 2.3. In vivo bioluminescence imaging been successfully employed in bioluminescence imaging stud- ies to monitor HSV-1 infection in vivo in both wild-type and In vivo bioluminescence imaging was performed using the knock-out mice [6,7,17]. Although this type of imaging cooled IVISÒ animal imaging system (Xenogen Corp., Ala- method potentially offers significant advantages over standard meda, CA, US) linked to a PC running Living ImageÔ molecular techniques, it has also a few disadvantages, e.g., the (Xenogen Corp.) and IGOR software (Wavemetrics, Seattle, attenuation of light by hair and organ pigmentation, overlap- WA, USA) under Windows XP. The IVIS system consists ping signals, the attenuation of signals due to organ depth of a cooled CCD camera mounted on a light-tight specimen from the surface, etc., but to date no comparison of these tech- chamber, a cryogenic refrigeration unit, a camera controller, niques has been made. The present paper reports the progres- and a computer system for data analysis. This system pro- sion of HSV-1 infection in mice as shown by this novel vides high signal-to-noise images of luciferase signals emit- bioluminescence technique, and compares bioluminescent ted from within living animals. The luciferase enzyme tracking of the virus with the well-established method of produces light in the presence of the substrate luciferin, oxy- real-time PCR. In vitro light emission in selected organs gen and ATP [19]; the light produced penetrates into 1332 J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338 mammalian tissues and can be externally detected and quan- 10 min. The image acquisition parameters were identical to tified using sensitive light-imaging systems [20]. Prior to im- those used for living animals. aging, D-luciferin (Xenogen Corp.) was prepared in normal saline as a 10-mM stock solution in phosphate-buffered sa- 2.5. HSV-1 DNA quantification in tissue homogenates line (PBS). The solution was sterile-filtered and stored in al- iquots at 80 C, and the mice were i.p.-injected with a dose DNA from homogenized samples was extracted by con- of 15 mg/kg body weight. D-luciferin is light-sensitive, so the ventional methods (High Pure PCR Template Preparation reagent and solution were adequately protected at all times. Kit, Cat. 1 796 828, Roche, Germany). The concentration Mice were anesthetized by i.p. injection with a dose of of HSV-1 DNA in different organs was then quantified by 0.02 ml/g body weight of 1.2% Avertin solution (2,2,2-tribro- real-time quantitative PCR in an ABI Prism 7900HT SD sys- moethanol and tert-amylalcohol, Aldrich). Anesthesia was ef- tem (Applied Biosystems) using a Custom Taqman assay (a fective after some 5 min and lasted for 25e30 min. Imaging specific assay for a sequence against the US12 viral gene). began 10 min after the administration of D-luciferin. A gray- Reactions were performed under universal conditions using scale surface image of each mouse was initially acquired us- the Taqman Universal PCR Master Mix (Applied Biosys- ing field-of-view position C, a 0.2-s time, a medium tems). Exactly the same PCR protocol was used to quantify binning (resolution), an f/stop of 8 () and an open fil- the mouse genomic DNA, using an Assay-on-Demand probe ter. An integration time of 10 min (for dorsal imaging) or specific for the GAPDH house-keeping gene (code 5 min (for ventral imaging) with large binning (high resolu- Mm99999915_g1; Applied Biosystems). An appropriate con- tion), an f/stop of 1 and an open filter were used for lumines- centration range of virus was used for the optimization of the cent image acquisition. The distance of the mice from the standard curve, and the viral DNA concentration was ex- CCD camera was always the same (position C). Photon pressed in terms of viral copy numbers. PCR calibration flux data were normalized for differences in the image acqui- was performed by amplification of the GAPDH house-keep- sition time. Signal intensity was quantified as the flux of all ing gene from a concentration range of mouse genomic the detected photon emissions within the region-of-interest DNA (results expressed as nanograms of DNA of GAPDH). (ROI) of the mouse body using Living ImageÔ software. Viral DNA loads were also corroborated by amplification of A group of 4 animals were evaluated at each time point; the thymidine kinase viral gene and the b-actin house-keep- a representative animal of each is shown in the corresponding ing gene by real-time PCR using a LightCycler rapid thermal figures. cycler (Roche Diagnostics Ltd, Lewes, UK) and a LightCycler FastStart DNA Master SYBR Green I kit (Cat. 3 003 230, Roche, Germany) as previously described [12e14,21]. Each 2.4. Quantification of bioluminescence data experiment was performed in triplicate. Melting curve analy- ses, agarose and acrylamide gel electrophoresis, restriction The gray-scale of the mice obtained in the spec- analysis and nested-PCR confirmed the specificity of the imen chamber under dim LED illumination was overlain with products. a pseudocolor luminescent image showing from violet (least intense) to red (most intense). The variation repre- 2.6. In vitro luciferase luminometer assay sents the spatial distribution of the photon emissions emerging from luciferase activity within the animal. Signal intensities The ovaries and adrenal glands of the mice used to quantify from manually derived ROIs were obtained over the dorsal HSV-1 DNA loads were also used to quantify in vitro light or ventral area of each animal, and the processing software emission. These organs were selected because they showed was used to quantify light emission from the . Data the highest viral DNA levels, and because the bioluminescence were expressed as photon flux (photons/s/cm2/steradian), images obtained suggested that these organs were major points where steradian (sr) refers to the photons emitted from a solid of infection. These tissues were homogenized in PBS in angle of a sphere. The background photon flux was defined a Mixer Mill Type MM 300 (RetscheQiagen, Hann, Ger- from an ROI of the same size (4.8 6.1) placed in the same many) and CCLR lysis buffer (Promega, Madison, WI, US) position prior to the injection of D-luciferin. These figures added. After freeze-thawing at e80 C for 15 min three times, were subtracted from the photon flux in each region to quan- the homogenate was centrifuged at 1870 g for 15 min. FL tify relative luciferase activity as a measure of the amount of activity was assessed using 20 ml of supernatant with 100 ml virus. For all acute infection experiments, a threshold value luciferase assay reagent (Promega, Madison, WI, US). A lu- of 10% light emission was set to distinguish true results minometer (Monolight 2010, Analytical Luminescence, San from the background. A manually defined ROI was then drawn Diego, CA, US) was used to measure total light emission ac- and all light emerging from it taken into account. Data were cording to the manufacturer’s protocol. The results were nor- normalized to the peak signal intensity of each time course malized to relative light units (RLU) per gram of protein as and reported as mean values standard deviations (SD). On measured by the BCA Protein Assay System (Pierce, Rock- day 7, when the bioluminescent signal was undetectable, ani- ford, US). Results from these in vitro assays were compared mals were sacrificed and bioluminescence measured in iso- with bioluminescence signals and viral DNA concentrations lated organs previously soaked in a D-luciferin-bath for obtained for all mice. J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338 1333

3. Results the animals up to day 4 post-infection. No luciferase activity was detected in mice on day 5.7, consistent with viral clear- 3.1. Clinical scores ance, its entry into the central nervous system and the estab- lishment of latency. The bioluminescence on the ventral area None of the mice showed any clinical symptoms of disease of the mice showed similar FL activity. Light emission was re- after inoculation with 2 107 PFU of KOS/Dlux/oriL HSV-1. stricted to regions compatible with the adrenal glands (Fig. 2, None of the mock-infected animals showed any clinical white arrows) and ovaries (asterisks), again demonstrating the abnormalities. extensive dissemination of the virus beyond the site of perito- neal infection. The photon flux decreased markedly in a time- dependent fashion in both the dorsal and ventral regions of the 3.2. Bioluminescence imaging of reporter virus mice; the photon flux from the head ROI remained essentially at background level throughout the course of infection. Photon The progression of acute infection was monitored until the flux quantification data are expressed as means SD (Fig. 3). disappearance of the signal, using the distribution and relative On day 7, when the bioluminescence had disappeared in the intensity of transmitted light to determine the sites of infection living animals, the latter were sacrificed and their organs were and relative amounts of replicating reporter virus (luciferase immediately removed. Isolated organs were placed in a d- protein would only be detected once the viral genome has luciferin bath and bioluminescence imaging was undertaken been translated). Serial images were obtained from all animals (Fig. 4). Signals were restricted to the spinal cord, although and the mean photon flux relative to the peak signal deter- the photon flux levels were low, confirming the data obtained mined. No bioluminescence was detected above background in intact animals. No bioluminescence was detected in levels in mock-infected mice or before the administration of the brain or any other excised organ. These results show the D-luciferin. Fig. 1 shows the bioluminescence images of a rep- final e but minimal e spread of the virus to the CNS during resentative infected animal over time and the signal variability infection. between mice at one selected time point (day 2). Each result is a pseudocolor illustration overlain on a gray-scale reference image of the whole mouse. 3.3. Detection and quantification of HSV-1 DNA In all the animals, the highest levels of luciferase activity were detected on the dorsum at day 0.8. Signals decreased To more precisely localize the KOS/Dlux/oriL virus, and gradually thereafter, remaining localized to the abdomen of given the problem that light emission is attenuated

Fig. 1. Time course of hematogenous infection with KOS/Dlux/oriL virus as shown by bioluminescence imaging. Four 9-week-old CD-1 female mice were i.p.-injected with a suspension of 2 107 PFU of recombinant HSV-1 and bioluminescent images superimposed on gray-scale of mice at 0.8, 2, 3, 4, 5.7, and 7 days post-infection (top). A representative animal of each time point group is shown (mouse 2). Variability between animals is represented by showing the four evaluated mice at day 2 post-infection (bottom). The relative levels of bioluminescence are shown as a pseudocolor display, with red and violet representing the strongest and weakest photon fluxes, respectively. 1334 J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338

Fig. 2. Bioluminescence imaging of the ventral area at 3 days post-infection Fig. 4. In situ localization of FL activity 7 days post-infection. Four 9-week- with KOS/Dlux/oriL virus. Four 9-week-old CD-1 female mice of were i.p.- old CD-1 female mice were i.p.-inoculated with a suspension of 2 107 PFU injected with a suspension of 2 107 PFU of recombinant HSV-1 and biolu- of KOS/Dlux/oriL HSV-1 and sacrificed at day 7 post-infection. Organs were minescent images superimposed on gray-scale photographs of mice at 3 day dissected out and placed separately in a D-luciferin-bath. Ten minutes later post-infection. A representative animal of the group of mice is shown. The bioluminescent images were taken and superimposed on gray-scale photo- relative levels of bioluminescence are shown as a pseudocolor display, with graphs. The representative image shows the distribution of FL activity in the red and violet representing the strongest and weakest photon fluxes, respec- specimen; the spinal cord was positive. tively. Asterisks show foci of infection compatible with the ovaries. White arrows indicate signals compatible with the adrenal glands. glands, spinal cord and mesenteric lymph nodes, while in the brain and trigeminal ganglia detectable HSV-1 DNA levels approximately 10-fold for every centimeter of tissue through were very significantly lower. The viral DNA concentration in which it passes [3], the luciferase activity recorded was com- the adrenal glands and ovaries peaked between day 2 and 3. pared to real-time PCR quantification of viral DNA in selected On day 3 HSV-1 DNA was cleared from the lymph nodes. organs. HSV-1 DNA concentrations showed a constant level of The spinal cord was the region of the nervous system with virus in the bloodstream independent of the time point (Fig. 5). the greatest viral DNA loads, peaking around day 4 when At all time points, the adrenal glands and ovaries were the or- the HSV-1 DNA concentrations in the adrenal glands dimin- gans that showed the highest viral DNA levels. These results ished. In the peripheral nervous system, HSV-1 DNA was con- agree with the bioluminescent signals detected in the abdomen stantly detected in the trigeminal ganglia until day 5.7, after using the IVIS system (Fig. 1). During the initial stages of in- which it was cleared. fection, HSV-1 DNA was detected in the ovaries, adrenal

3.4. Correlation of in vivo bioluminescence imaging with HSV-1 DNA quantification

In general, the PCR-quantified viral DNA levels reproduced the in vivo imaging results. Both reflected the known progres- sion of acute hematogenous HSV-1 infection [12e14], al- though no brain colonization was seen with this recombinant strain. Fig. 6 shows the quantification of viral genomes over time in three categories of organs as follows: (i) total organs (corresponding to the sum of HSV-1 DNA concentrations from all organs), (ii) the peripheral organs (the sum of HSV-1 DNA concentrations for the whole blood, mesenteric lymph nodes, adrenal glands and ovaries), and (iii) the nervous sys- Fig. 3. Quantification of the in vivo luciferase signal of the dorsal area during tem (the sum of HSV-1 DNA concentrations for the spinal the time course of infection. Four 9-week-old CD-1 female mice were i.p.- cord, trigeminal ganglia and brain). The contribution of the pe- inoculated with a suspension of 2 107 PFU of KOS/Dlux/OriL HSV-1 and ripheral organs almost entirely accounted for the total organs’ luminescent images reordered at 0.8, 2, 3, 4, 5.7, and 7 days post-infection value; the nervous system contributed very little. to monitor the progression of infection. After applying a minimum threshold value to the image, an ROI was manually defined. Data are plotted as the Some small variations were seen between the biolumines- mean and standard deviations of photon counts over time from four animals cence imaging and real-time PCR results. Whereas the maxi- per group. mum bioluminescent signal was observed on day 0.8 (Fig. 3), J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338 1335

Fig. 5. Time course of KOS/Dlux/oriL HSV-1 infection by quantification of HSV-1 DNA in a number of analyzed organs. Thirty-two 9-week-old female mice were i.p.-inoculated with a suspension of 2 107 PFU of HSV-1 (KOS/Dlux/oriL), sacrificed at several time points (ranging from 0.8 to 7 days post-infection [dpi]), and dissected. The lines represent the viral copy numbers detected in each organ expressed on a logarithmic scale. Values are the mean SEM of the quantity of viral genomes normalized with respect to the number of mouse genomes expressed in 100 ng of host DNA by amplifying the GAPDH house-keeping gene. the HSV-1 DNA peak was reached on day 2. These discrep- screened in the ovaries and adrenal glands (Fig. 7). The stron- ancies might be explained in that the PCR detects viral gest light intensity was generated on day 2 in both organs, be- genomes while bioluminescence imaging tracks replicating coming significantly reduced on each subsequent day, and virus. Moreover, since the bioluminescence images are two- undetectable from day 4. The ovaries showed stronger in vitro dimensional, light from the abdomen on dorsal images might photon emission than the adrenal glands, confirming the data arise from more than one superimposed or adjacent organs obtained by in vivo bioluminescence imaging (Fig. 2) and or tissues, with relatively greater light emissions detected at from HSV-1 DNA determinations (Fig. 5). These results sug- sites closer to the CCD camera. However, the correlation gest that real-time PCR viral DNA quantification strongly cor- between both techniques was good, and the photon flux deter- relates with photon flux values, both in vivo and in vitro. mined by ROI analysis correlated strongly with input virus titers of over 104 viral genomes per nanogram of host DNA 4. Discussion at all time points. The progression of infection, as determined by in vivo imaging and real-time PCR, suggested the follow- These results show that a recombinant KOS strain, HSV-1 ing route for KOS/Dlux/oriL infection: blood, ovaries and virus (KOS/Dlux/oriL) expressing FL driven by early gene adrenal glands, and finally spinal cord. promoters [6], allows non-invasive bioluminescence imaging to be used in the studies of hematogenous HSV-1 infection 3.5. Correlation between in vivo bioluminescence, HSV-1 in living mice. The replication and spread of KOS/Dlux/oriL DNA quantification data, and in vitro light emission in the mouse ocular infection model are reported not to differ significantly from that of the wild-type KOS strain, validating To confirm the acute infection data obtained by in vivo light this reporter virus for the real-time study of HSV-1 pathogen- emission and real-time PCR, the in vitro activity of FL was esis [7]. However, until now there has been no published 1336 J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338

[2,24]. However, this method also has a number of limitations as follows: light transmission is attenuated by hair and organ pigmentation, photon flux from a superficial infection will be greater than that for the same titer of virus in an internal organ, bioluminescence may arise from overlapping sites of viral infection (thus overestimating the signal), spatial resolu- tion of 2e3 mm distinguishing between adjacent anatomical sites may not be feasible regardless of the image matrix, and light is attenuated approximately 10-fold for every centimeter of tissue through which it passes [3]. Moreover, image acqui- sition time is empirically determined based on anticipated amounts of luciferase activity [23], thus, for low amounts of bioluminescence, long imaging times may be needed to detect luciferase activity and to optimize the signal-to-noise ratio [5]. Complementing with conventional molecular techniques such as real-time PCR or luminometer assays therefore appears to be necessary. Fig. 6. Quantification of HSV-1 DNA during the time course of infection in several organ groups. Thirty-two 9-week-old female mice were i.p.-inoculated In previous work we characterized hematogenous HSV-1 7 e with a suspension of 2 10 PFU of HSV-1 (KOS/Dlux/oriL), sacrificed at infection using the KOS [12 14] and F strains (manuscript several time points (ranging from 0.8 to 7 days post-infection [dpi]) and dis- submitted), and showed the importance of viremia, the ex- sected. The bars represent the viral copy numbers for each group of organs ex- treme susceptibility of the adrenal gland and the ovary, and pressed on a logarithmic scale as follows: (i) total organs (corresponding to the the neurotropism of HSV-1 in such infections [12e14]. The sum of the viral DNA concentrations for the whole blood, mesenteric lymph nodes, adrenal glands, ovaries, spinal cord, trigeminal ganglia and brain), present real-time PCR results show that, after i.p. injection (ii) peripheral organs (sum of whole blood, mesenteric lymph nodes, adrenal with KOS/Dlux/oriL HSV-1, the virus is immediately detect- glands and ovaries), and (iii) nervous system (sum of spinal cord, trigeminal able in blood, replicates in the adrenal gland and ovary, and ganglia and brain). Values are the mean SEM of the number of viral ge- eventually reaches the nervous system. The mesenteric lymph nomes normalized with respect to the quantity of mouse genomes expressed nodes are also permissive to infection, but the virus does not in 100 ng of host DNA by amplifying the GAPDH house-keeping gene. appear to replicate in them, as previously suggested [25]. The bioluminescence results obtained confirm the importance report of the time course of hematogenous infection with of the ovaries and adrenal glands in hematogenous infection, KOS/Dlux/oriL virus. and the connection with the spinal cord. Although the present Non-invasive imaging with D-luciferin (which is of low im- HSV-1 DNA concentrations in the ovaries and adrenal glands munogenicity and negligible toxicity) provides advantages mimicked our previous data for the wild-type KOS strain in- such as the ability to detect infection at any site in the living jection [12,14], the neurotropic character of this recombinant mouse [4,22,23], the possibility of repeat imaging of the virus was considerably less obvious. In fact, KOS/Dlux/oriL same mouse, and the easy quantification of bioluminescence virus was detected only in the spinal cord and only after the animals had been sacrificed. However, the general behavior of infection at the bloodstream level was similar, indicating that this is a general and reproducible phenomenon, and that recombinant KOS/Dlux/oriL is a valid reporter virus. An important conclusion to be drawn from the present stud- y is that changes in viral DNA concentration correlate with differences in the photon flux values, both in vivo and in vitro. However, the light produced by KOS/Dlux/oriL in organs close to one another could not be properly distinguished. This was solved by using real-time PCR or luminometer as- says after the sacrifice of the mice. The PCR technique showed the peak HSV-1 DNA loads in the adrenal glands and ovaries appear on day 2e3 after infection. However, the strongest sig- Fig. 7. Time courses of FL activity during KOS/Dlux/oriL infection in the nal recorded in bioluminescence imaging corresponded to day ovaries and adrenal glands. Thirty-two 9-week-old female mice were i.p.- 0.8 (over the peritoneal cavity and on dorsum); by day 4 post- inoculated with a suspension of 2 x 107 PFU of HSV-1 (KOS/Dlux/oriL), infection, the signal in all mice was at or close to background sacrificed at several time points (ranging from 0.8 to 7 days post-infection levels. The minimal differences in the results provided by [dpi]), and dissected. In vitro light emission was quantified by luminometer these techniques may result in very slightly different viral pro- assays as detailed in Section 2. The values are the mean SEM of the relative light units (RLU) of FL activity normalized with respect to the protein concen- gression patterns. It should also be remembered that the pres- tration expressed in grams. The solid lines represent the viral loads detected in ence of luciferase activity indicates viral replication rather ovaries; the dotted lines show the viral loads in the adrenal glands. than the simple presence of free viral particles. In addition J.S. Burgos et al. / Microbes and Infection 8 (2006) 1330e1338 1337 luciferase requires ATP to produce bioluminescence from lu- Universidad Auto´noma de Madrid for supporting our research ciferin [26], so extracellular virus cannot be detected [27]. and the Fundacio´n Areces for an institutional grant awarded to PCR, however, detects every viral genome, both inside and the Centro de Biologı´a Molecular Severo Ochoa. This work outside of cells. Nevertheless, both the viral DNA levels and was also supported by grant (SAF-2002-04122-C03-02) from the imaging data revealed similar KOS/Dlux/oriL infection the Spanish Ministry of Science and Technology. We thank profiles, and it has previously been demonstrated that the rel- Prof. Federico Mayor for his continuous encouragement and ative differences in virus titer at certain sites correlate with the help, and Dr. David A. Leib for providing the KOS/Dlux/ bioluminescence measured in ROI analysis [7]. oriL HSV-1 and for his critical reading of the article. We One of the main drawbacks of in vivo bioluminescence imag- also thank Dr. Marı´a J. Bullido for helping with virus quanti- ing is the lack of detailed tomographic information. As a result, fication using the ABI Prism 7900HT SD system (manuscript one cannot be certain which organ or tissue has the greatest FL in preparation). We gratefully acknowledge Meritxell Huch for activity. Given the direct relationship between the amounts of in- her help at the Centre de Regulacio´ Geno`mica. fectious virus and light emission, luminometer assays were also used in the present study to monitor HSV-1 hematogenous infec- References tion. 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