Fig. 7
From: Chemokines cooperate with TNF to provide protective anti-viral immunity and to enhance inflammation
The activity of the SECRET domain in vivo is dependent on concomitant TNF blockade. a Schematic diagram of the genomic structure of ECTV RevSECRET. The genes flanking the CrmD locus are indicated. In gray, the CrmD locus in the parental and recombinant virus is shown. Presence of a point mutation (N77F) in the CrmD of ECTVRevSECRET is indicated by an asterisk and a black line. Only the left inverted terminal repeat is shown. b Western blot analyses using anti-CrmD and anti-35-kDa vCKBP antisera of supernatants from BSC-1 cells mock-infected (1) or infected with ECTVΔCrmD (2), ECTVRevCrmD (3), ECTVRevCRD (4) or ECTVRevSECRET (5) at a multiplicity of infection of 5 PFU/cell and harvested at 24 h post-infection. The position of the proteins is indicated by arrows. Molecular size markers in kDa are shown on the left. c TNF-induced cytotoxicity assay. Increasing amounts of supernatants (equivalent to 2, 8 or 16 × 103 cells) from cells infected with the indicated viruses were added to block the effect of TNF on L929 cells. Values of % cell viability obtained in the presence of mock-infected cell supernatants were subtracted from the % cell viability caused by the corresponding supernatant volume from virus-infected cells. Data are mean +/− SD of triplicate samples. d Mortality rate determination of CrmD recombinant ECTVs in susceptible mice. Groups of five or ten female BALB/c mice were infected s.c. in the left hind footpad with different doses of the indicated viruses. The number of survivors at 15 dpi and the mean time to death (MTD) in days for each condition are shown. n.a., not applicable. e Groups of five female BALB/c mice were inoculated s.c. in the left hind footpad with 106 PFU of ECTV RevCrmD (black circles), ECTVΔCrmD (black triangles), ECTVRevCRD (white triangles) or ECTVRevSECRET (white squares) and monitored daily for mortality and footpad swelling, as indicated. Data are shown as mean +/− SD. Asterisks indicate time points at which significant differences (p < 0.01, multiple t tests with false discovery rate correction at Q = 1%) between ECTVΔCrmD and ECTVRevSECRET-inoculated mice were found. f Replication of recombinant ECTVs in spleen. Viral titres at 7 dpi in spleen of mice infected with 105 PFU per animal of the indicated viruses. Data are mean log +/− SD of groups of five mice for each condition. Groups significantly different from the ECTVRevCrmD-infected group are indicated (asterisks p < 0.05, ANOVA with Bonferroni multiple comparison test)
