In vivo imaging of murid herpesvirus-4 infection

Abstract

Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7-10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions.

Fig. 1.

Generation of MuHV-4 expressing luciferase. (a) A 2 kb luciferase-polyA cassette was placed downstream of a 500 bp M3 promoter, in a MfeI restriction site between ORFs 57 and 58. Relevant restrictions sites are shown. (b) Viral DNA was digested with EcoRI or HindIII and probed with the 75 338–78 717 BglII clone shown in (a). The luciferase expression cassette changes a 14.9 kb EcoRI band to 5.5 kb+12.0 kb, and a 14.5 kb HindIII band to 6.8 kb+10.1 kb. M3-LUC1.6 and M3-LUC2.1 are independently generated recombinant viruses. (c) BHK-21 cells were left uninfected or infected overnight (1 p.f.u. cell⁻¹), then lysed and assayed for luciferase expression. Each bar shows the mean±sd of triplicate cultures. (d) BHK-21 cells were infected with wild-type or M3-LUC MuHV-4 (0.01 p.f.u. cell⁻¹, 2 h), washed with PBS to remove unbound virions, then incubated at 37 °C. The infectious virus in each culture was measured by plaque assay. (e) BHK-21 cells were infected with wild-type MuHV-4, the M3-LUC2.1 recombinant or its ORF50⁻ derivative. Luciferase expression was assayed 18 h later by luminometry. Each bar shows mean±sd of five replicate infections. The ORF50⁻ cultures contained no replication-competent virus by plaque assay.

Fig. 2.

In vivo infection by luciferase-expressing MuHV-4. (a) M3-LUC1.6 and M3-LUC2.1 were compared with wild-type MuHV-4 for their capacity to colonize mice after intranasal infection. Lytic replication was tested by plaque assay of lungs after 5 days. Latency establishment was tested by infectious centre assay of spleens after 13 days. Each point shows the titre of one mouse. There was no significant difference between each virus. (b) Mice were infected intranasally (10⁴ p.f.u.) with M3-LUC2.1 MuHV-4 under general anaesthesia, and then injected with luciferin and imaged every 3–4 days. Images show a representative mouse at day 7 and 14 p.i. The signal in the mouth was atypical and probably corresponds to the strong neck signal reflecting off the incisors. The scale bar shows photons s⁻¹ cm⁻² sr⁻¹.

Fig. 3.

Luciferase signals from isolated organs after intranasal MuHV-4 infection. Mice equivalent to those in Fig. 2(b) were dissected and their organs imaged ex vivo. Each image is representative of data from at least five mice, and shows either a standard photograph (Photo) or that photograph overlaid with the luciferase signal (Photo+LUC). The colour scheme for relative signal intensity is as for Fig. 2.

Fig. 4.

Quantification of luciferase signals from isolated organs after intranasal MuHV-4 infection. Mice were infected intranasally (10⁴ p.f.u.) with M3-LUC. At each time point, at least five mice per group were dissected for ex vivo organ imaging. Each point shows the maximum radiance value for one mouse. The horizontal dashed lines mark an arbitrary sensitivity threshold, chosen to minimize the chance of artefactual signals such as secondary light reflections.

Fig. 5.

Comparison of intranasal and intraperitoneal MuHV-4 infections. (a) Mice were inoculated intranasally or intraperitoneally with 10⁴ p.f.u. of M3-LUC, then monitored for luciferase expression. Representative pairs of mice are shown. (b) Mice were dissected at 4 or 10 days after intraperitoneal virus inoculation to identify the source of the live imaging signals. The colour scheme for relative signal intensity is as for Fig. 2.

Fig. 6.

High dose and low dose intranasal infections. Mice were infected intranasally with M3-LUC (10² or 10⁵ p.f.u.) under general anaesthesia, then live-imaged for luciferase expression every 3–5 days. Each point corresponds to one mouse. The horizontal dashed lines mark an arbitrary sensitivity threshold.

Fig. 7.

Intranasal infection with and without anaesthesia. (a) Mice were infected intranasally with M3-LUC (10⁴ p.f.u.), either awake or while anaesthetized, then imaged for luciferase expression every 3–5 days. Each point corresponds to one mouse. The horizontal dashed lines mark an arbitrary sensitivity threshold. (b) Representative images from the data summarized in (a). (c) At 1 month p.i., mice were analysed for viral genome loads in the spleen and SCLN by real-time PCR. The top panel shows the cellular control (APRT), the middle panel the MuHV-4 M2 gene, and the bottom panel the M2 load normalized by APRT. Each point corresponds to one mouse. There was no statistically significant difference between mice given anaesthesia (Inf. lung) or not (Inf. nose).

Fig. 8.

Oral infection. (a) Representative image of a luciferase-positive mouse 8 days after oral infection under general anaesthesia. (b) Representative image of a luciferase-positive mouse 7 days after oral infection without anaesthesia. (c) Sera from mice 1 month after oral inoculation without anaesthesia (50 p.f.u.) were analysed for MuHV-4-specific IgG by ELISA, along with pooled naive mouse serum, pooled immune serum and serum from a mouse infected (10 p.f.u.) intranasally without anaesthesia.