Microbes and Infection 8 (2006) 1330e1338 www.elsevier.com/locate/micinf Original article Non-invasive bioluminescence 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 luciferase 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 light 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 camera; 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 Â 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.