J. S. May and P. G. Stevenson Fig. 5. Protection of naive mice and induction of MuHV-4 neutralization by vaccination with a gHL fusion protein. (a) 129Sv C57BL/6 FcRc”/”FccRII”/” mice (FcR”/”) or 129Sv controls were primed i.p. with vaccinia virus recombinants (105 p.f.u.) and 1 month later challenged i.n. with MuHV-4 (104 p.f.u.). Infectious virus titres in lungs and noses were measured by plaque assay 6 days later. Each point shows the result for one mouse. The bars show geometric means. VAC-gB significantly reduced virus replication in lungs (P,0.00001) and noses (P,0.04) of 129SV but not FcR”/” mice. (b) Sera were pooled from mice (n510) 3 months after i.p. infection with VAC-gB-N or VAC-cont, then given i.p. to 129Sv or FcR”/” mice (500 ml per mouse) at the same time as i.n. MuHV-4 (5000 p.f.u.). Lungs were titrated for infectious virus by plaque assay 5 days later. Each point shows the result for one mouse, and the bars show geometric means. gB-N-immune sera significantly reduced virus titres in 129Sv mice (P,0.0001 by Student’s two-tailed t-test) but not in FcR”/” mice (P50.86). CHO-gB-N cells, the coding sequence for gB N-terminal to its furin cleavage site (aa 1–423) was cloned upstream of a GPI anchor attachment site as for full-length gB (Lopes et al., 2004). CHO-K1 cells transfected with the gB-N expression plasmid were selected with hygromycin (250 mg ml21). Viruses. All MuHV-4 derivatives were generated from a BAC-cloned genome (Adler et al., 2000). Luciferase expression from an ectopic MuHV-4 lytic promoter has been described previously (Milho et al., 2009). The MuHV-4 BAC contains an HCMV IE1 promoter-driven eGFP expression cassette. However, this is poorly transcribed in some cell types (Rosa et al., 2007). Therefore to assay in vitro infection, we inserted into the MuHV-4 genome a separate, intergenic eGFP expression cassette with an EF1a promoter. We first mutated the internal BglII site of the EF1a promoter in pBRAD from AGATCT to AGGTCT by overlap PCR, then PCR-amplified the modified promoter, adding BglII and EcoRI sites to its respective 59 and 39 ends, and cloned upstream of a poly(A) site in pSP73 (May et al., 2005). The eGFP coding sequence was PCR-amplified from pEGFP-N3 (Clontech), adding 59 EcoRI and 39 XhoI sites, and cloned into EcoRI/SalI sites between the EF1a promoter and poly(A) site. The resulting eGFP expression cassette was then subcloned as a blunted BglII–XhoI fragment into the MfeI site of a 3.4 kb BglII genomic clone, between the 39 ends of ORFs 57 and 58 (May et al., 2005). Inserts here have no obvious effect on MuHV-4 replication (Bennett et al., 2005). The eGFP expression cassette and its genomic flanks were then subcloned into the pST76K-SR shuttle vector and recombined into the MuHV-4 BAC by standard methods (Adler et al., 2000). Recombinant clones were identified and checked for genome integrity by restriction enzyme mapping. Infectious virus was recovered by transfecting BAC DNA into BHK-21 cells. The BAC cassette was then removed by passage through NIH-3T3-CRE cells. Virus stocks were grown in BHK-21 cells. Virions were harvested from infected cell supernatants by ultracentrifugation, and cell debris removed by low speed centrifugation (de Lima et al., 2004). 2548 Vaccinia viruses expressing a GPI-linked gB extracellular domain or gH/gL fusion protein have been described previously (Gillet et al., 2007b). To make VAC-gB-N, the coding sequence for gB aa 81–423 was amplified by PCR, digested with XbaI at a site internal to gB and cloned as a blunt XbaI fragment into the SnaBI/XbaI sites of gB in pBRAD-gB. The predicted gB signal sequence is aa 1–24, and SnaBI cuts after residue 22, so the coding sequence for residues 23–25 was included in the 59 PCR primer, thereby reconstituting the native signal sequence without residues 26–80. The modified gB-N coding sequence plus C-terminal GPI attachment site was PCR-amplified from pBRAD-gB-N using 59 NheI-restricted and 39 HindIII-restricted primers and cloned into the NheI/HindIII sites of the vaccinia recombination plasmid pMJ601 (Davison & Moss, 1990). pMJ-601- gB-N-GPI was then transfected into vaccinia virus-infected TK2143 cells. Thymidine kinase-deficient recombinants were selected by passage in 25 mg 59-bromo-29-deoxyuridine (Sigma Chemical Co.) ml21, identified by b-galactosidase expression using X-Gal, and plaque purified. Virus stocks were grown and titrated using TK2143 cells. As a TK2 control, we used vaccinia virus expressing the mouse invariant chain coding sequence with ovalbumin residues 323–339 substituted for the CLIP peptide (Smith et al., 2006). Virus titres. Virus stocks and ex vivo samples were titrated for infectivity by plaque assay on BHK-21 cells (de Lima et al., 2004). Lungs and noses were removed from mice post-mortem, freeze– thawed, then homogenized in DMEM. Nose samples included the turbinates and nasal septum, which contain all the nasal luciferase signal of mice infected with luciferase+ MuHV-4 (Milho et al., 2009). Serial dilutions of each sample were incubated (2 h, 37 uC) with BHK-21 cell monolayers, then overlaid with DMEM plus 0.3 % carboxymethylcellulose. After 4 days the monolayers were fixed in 4 % formaldehyde, stained with 0.1 % toluidine blue and plaques were counted with a plate microscope. Neutralization assays. EF1a-eGFP MuHV-4 was incubated with serum dilutions (2 h, 37 uC), then added to BHK-21 fibroblasts or Journal of General Virology 91