Kinetics of Mx expression in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar L.) parr in response to VHS-DNA vaccination

The duration of the Mx mRNA response to an intramuscular injection of the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) gene DNA vaccine as well as to the control plasmid was determined in rainbow trout at 14 degrees C over a period of 11 weeks. The Mx response was detectable on day 7, peaked on day 14 and returned to pretreatment levels on day 21 and thereafter. No increase in Mx expression was detectable to the control plasmid. In further experiments, the kinetics of the Mx response were compared in rainbow trout and Atlantic salmon parr kept at 10 degrees C and injected with the DNA vaccine or the synthetic double-stranded RNA, poly I:C. In both species there was a rapid response to poly I:C detectable from day 1, reaching maximum from days 3 to 9 and decreasing to background level by day 12. The peak level and return to background was reached slightly later in salmon. In both species the response to the VHS/DNA vaccine was slower to begin, not being detectable on days 1 and 3, but elevated levels were found on day 6. However, in the salmon parr, the peak level was on day 6 and the signal disappeared by day 12, while in the rainbow trout, the response peaked at day 12 and lasted until day 21. The kinetics of the Mx response to the VHS/DNA vaccine in rainbow trout correlate with the early non-specific protection against VHS in this species following vaccination. It is speculated that the more transient Mx response in Atlantic salmon parr to the DNA vaccine may be related to the innate resistance of salmon to VHS.     

AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.     

Endocytosis plays a critical role in proteolytic processing of the Hendra virus fusion protein

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F(0), which is proteolytically processed to the mature form, F(1) + F(2). Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca(2+), and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXPhi. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.     

Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt alpha-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion.     

Requirements for estrogen receptor alpha membrane localization and function

The estrogen receptor alpha (ERalpha) exists as a functional receptor at the plasma membrane. The structural requirements for localization and function are not well understood. Several laboratories have recently elucidated certain requirements. We recently found the translocation of ERalpha to the membrane in the absence of estrogen is dependent on caveolin-1 and serine 522 of the ERalpha protein. Mutation of serine 522 to alanine results in a 62% decrease in membrane localization and association with caveolin-1. Similarly, deletion of the caveolin-1 scaffolding domain (amino acids 60-100) largely prevents the localization of ERalpha at the plasma membrane. In the presence of estradiol (E2), ERalpha, Src-homology and collagen homology (Shc), and insulin-like growth factor receptor-1 proteins associate with and increase the localization of ERalpha at the membrane. Membrane-localized ERalpha functions as an atypical G-protein coupled receptor. There is no good evidence that ERalpha spans the membrane or contains an extracellular domain. E2/ERalpha activates different G-proteins in cell context-related fashion. These G-proteins lead to the activation of Src through PLC, PKC, IP3 and calcium influx. In breast cancer, Src activates matrix metalloproteinase-2 and -9, which cleaves heparin binding epidermal growth factor, and thus activates EGFR. This leads to downstream signaling through ERK and PI3 kinase, imparting cell growth and survival.     

A comparison of scar revision with the free electron and carbon dioxide resurfacing lasers

Laser scar revision was studied to measure the effects of targeting extracellular matrix protein versus tissue water on scar revision. We compared the free electron laser used at 7.7 microm (the amide III protein absorption band) to the carbon dioxide (CO2) laser and dermabrasion.Nude mice (n = 40) that had rejected skin grafts on their dorsal surface and developed mature scars were used as a model for scar revision. One-half of each scar was revised with either the free electron laser at 7.7 microm (32 to 38 mJ, nonoverlapping pulses delivered with a computerized adjustable pattern generator at 30 Hz, and two to three passes), a 100-microsec CO2 resurfacing laser (500 mJ, 5.0 Hz, and two to five passes), or dermabrasion. The untreated portion of each scar served as an internal control. Evaluation was by measurement of the clinical size of the scar using photography with quantitative computer image analysis to compare the data and histology to evaluate the quality and depth of the scars.The free electron laser at 7.7 microm was significantly better than the CO2 laser and dermabrasion for scar size reduction (p < 0.046 and p < 0.018). The CO2 laser and a highly skilled dermabrader were not statistically significantly different (p < 0.44). The result seen with less skilled dermabraders was significantly worse than all other methods (p < 0.009).The free electron laser at 7.7 microm, which is preferentially absorbed by the proteins of the extracellular matrix, provided better scar reduction than the CO2 resurfacing laser and dermabrasion. Dermabrasion by a skilled operator resulted in improvement similar to the results obtained with the CO2 resurfacing laser, but less skilled operators had significantly poorer results.     

Isolation of salmon pancreas disease virus (SPDV) in cell culture and its ability to protect against infection by the 'wild-type' agent

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15 degrees C. After 7-9 days post-inoculation, CHSE-214 cell supernatants contained between 10(7)-10(5) TCID50 ml(-1) The cultured isolate is chloroform- and pH 3.0-sensitive, and virions are 50-60 nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3-6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.     

Immune status: normal expression of MHC class I in the placenta and what is expected in clones

Major histocompatibility complex (MHC) class I genes are highly polymorphic, widely expressed genes that play a vital role in immune responses. Because of their involvement in tissue transplant rejection there has been much interest in their expression during pregnancy. This review examines the evidence for their role in livestock reproduction, and speculates that the process of creating clones may modify their expression with deleterious results.