The equine herpesvirus 1 UL34 gene product is involved in an early step in virus egress and can be efficiently replaced by a UL34-GFP fusion protein

The structure and function of the equine herpesvirus type 1 (EHV-1) UL34 homologous protein were characterized. A UL34 protein-specific antiserum reacted with an M(r)28,000 protein that could not be detected in purified extracellular virions. Confocal laser scanning microscopy demonstrated that UL34 reactivity mainly concentrated at the nuclear rim, which changed into a punctuate and filamentous pattern at late times after infection. These changes in UL34 distribution were especially prominent when analyzing the distribution of a GFP-UL34 fusion protein. A UL34-negative EHV-1 was generated by mutagenesis of a recently established BAC clone of EHV-1 strain RacH (pRacH). Release of extracellular infectious virus was severely impaired after infection of Rk13 cells with HDelta34. Electron microscopy revealed a virtual absence of virus particles in the cytoplasm of infected cells, whereas nucleocapsid formation and maturation within the nucleus appeared unaffected. A UL34-GFP fusion protein with GFP linked to the C-terminus of UL34 was able to complement for the UL34 deletion in trans, while a GFP-UL34-fusion protein with GFP linked to the N-terminus of UL34 was able to only partially restore virus growth. It was concluded that the EHV-1 UL34 product is essential for an early step in virus egress, i.e., release of capsids from infected-cell nuclei.     

The early UL3 gene of equine herpesvirus-1 encodes a tegument protein not essential for replication or virulence in the mouse

The UL3 gene of equine herpesvirus-1 (EHV-1) is retained in the genome of defective interfering particles and encodes a ~ 33 kDa myristylated protein. Further characterization showed that the UL3 gene is trans-activated only by the sole immediate early (IE) protein and encodes an early protein that is dispensable for EHV-1 replication and localizes in the tegument of purified virions. UL3-deleted EHV-1 (vL11ΔUL3) exhibits properties of host cell tropism, plaque size, and growth kinetics similar to those of the parental virus. Expression levels of EHV-1 proteins representative of all three gene classes in vL11ΔUL3-infected cells were identical to those in cells infected with parental virus. Mice intranasally infected with vL11ΔUL3 and parental virus showed no significant difference in mortality or virus lung titers. These findings suggest that the UL3 protein does not play a major role in the biology of EHV-1 in cell culture or virulence in the mouse.     

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2K) murine model

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a β-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.     

Transactivation by Runt related factor-2 of matrix metalloproteinase-13 in astrocytes

We have previously shown functional expression by osteoblasts of signaling machineries required for neurotransmission in the brain. In this study, we have evaluated possible functional expression of different osseous genes in the brain. In embryonic and adult mouse brains, mRNA expression was invariably seen for the master regulator of osteoblastic differentiation Runt related factor-2 (Runx2), in addition to the partner protein core binding factor-β and their targets such as osteopontin (OPN) and matrix metalloproteinase-13 (MMP13), but not for collagen-I or osteocalcin. In pluripotent P19 progenitor cells, Runx2 mRNA expression was drastically increased along with mRNA expression of an astrocytic marker, but not with neuronal marker mRNA expression. Both mRNA and corresponding protein were detected for Runx2 in cultured rat neocortical astrocytes and astrocytic C6 glioma cells. In C6 glioma cells, transient overexpression of Runx2 significantly increased mRNA expression of MMP13, but not of OPN. Moreover, transient overexpression of Runx2 significantly increased luciferase activity in C6 glioma cells transfected with the reporter plasmid linked to a wild-type Runx2 binding element in the MMP13 promoter, but not in cells with a mutated element. These results suggest that Runx2 signal input may lead to transactivation of MMP13 gene without affecting OPN expression in astrocytes.     

MIG (CXCL9) is a more sensitive measure than IFN-gamma of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines

For many years the IFN-γ ex vivo ELISPOT has been a major assay for assessing human T-cell responses generated by malaria vaccines. The ELISPOT assay is a sensitive assay, but an imperfect correlate of protection against malaria. Monokine induced by gamma (MIG), or CXCL9, is a chemokine induced by IFN-γ and has the potential to provide amplification of the IFN-γ signal. MIG secretion could provide a measure of bio-active IFN-γ and a functional IFN-γ signalling pathway. We report that detecting MIG by flow cytometry and by RT-PCR can be more sensitive than the detection of IFN-γ using these methods. We also find that there is little inter-individual variability in MIG secretion when detected by flow cytometry and that the MIG assay may be used to estimate the amount of bio-active IFN-γ present. Measurement of MIG alongside IFN-γ may provide a fuller picture of Th1 type responses post-vaccination.