Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Mole Ratio Dependence of the Mutual Deliquescence Relative Humidity of Aqueous Salts of Atmospheric Importance.

The response of the mutual deliquescence relative humidity (MDRH) of several mixed salt systems to changes in mole ratio is presented here. The MDRH values of NH4Cl-NaCl, NH4Cl-(NH4)2SO4, and, for the first time, the NaCl-NaBr systems were acquired as a function of mole ratio. These changes were studied using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The MDRH values of 1:1 salt mixtures were consistently found to be lower than the values of the individual deliquescence relative humidity (iDRH) of NH4Cl-NaCl and NH4Cl-(NH4)2SO4. The exception was the MDRH of the NaCl-NaBr system, which was found to be higher than the iDRH of NaBr particles, but lower than the iDRH of NaCl particles. When the mole ratio of the mixed system was varied, the MDRH of the mixtures showed a slight dependence on the mole ratio.

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007.

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Facing the threat of equine influenza.

Despite the availability of vaccines, equine influenza virus (EIV) continues to pose a threat to the racing industry. The virus spreads rapidly in unprotected populations and large scale outbreaks, such as those in South Africa in 2003 and Australia in 2007, can cost billions of pounds. Like other influenza viruses, EIV undergoes antigenic variation, enabling it to evade antibodies generated against previous infection or vaccination. The UK has an active surveillance programme to monitor antigenic drift and participates in an international collaboration with other countries in Europe, Japan and the USA to select suitable vaccine strains. Selection is primarily based upon characterisation of the viral haemagglutinin (HA), the surface protein that induces a protective antibody response; this protein is an important component of commercial vaccines. In recent years vaccine technology has improved and diagnostic methods have become increasingly sensitive, both play a crucial part in facilitating the international movement of horses. Mathematical modelling techniques have been applied to study the risk factors involved in outbreaks and provide valuable information about the impact of vaccination. Other factors, such as pathogenicity, are poorly understood for EIV yet may play an important role in the spread of a particular virus. They may also affect the ability of the virus to cross the species barrier, as seen with the transfer to dogs in the USA. Severity of infection is likely to be influenced by more than one gene, but differences in the NS1 protein are believed to influence the cytokine response in the horse and have been manipulated to produce potential vaccine strains.

Efficacy of a whole inactivated EI vaccine against a recent EIV outbreak isolate and comparative detection of virus shedding.

An outbreak of H3N8 Equine Influenza virus (EIV) that occurred in vaccinated horses in Japan was caused by a genetically divergent EIV isolate of the Florida clade 1 sub-lineage. This virus subsequently entered Australia where it infected thousands of immunologically naïve horses. The objective of this study was to evaluate the ability of a non-updated whole inactivated equine influenza (EI) vaccine to protect if used in the face of an outbreak induced by a virus similar to the ones circulating in Japan and Australia in 2007. Seven naïve Welsh mountain ponies were immunised twice with the commercially available vaccine Duvaxyn IE-T Plus and experimentally infected with A/eq2/Sydney/2888-8/07. Five ponies remained unvaccinated as controls. The ponies were challenged in an ACDP (Advisory Committee on Dangerous Pathogens) Category III containment facility by exposure to a nebulised aerosol of A/eq2/Sydney/2888-8/07 two weeks after the second vaccination. Clinical signs and virus shedding were monitored for 14 days post-challenge infection. After challenge infection, all control ponies developed clinical signs of disease with coughing being particularly noteworthy when compared with vaccinated ponies. Only 3 out of 5 controls developed pyrexia for up to 3 days, and 1 out of 7 vaccinates was pyretic for 1 day. Nasal discharge was evident in both control and vaccinated ponies with no significant difference between groups. Three different methods were used to measure virus shedding in nasal secretions (i.e. titration in embryonated hens' eggs, EIV NP ELISA and EIV NP qRT-PCR). The intensity and duration of EIV shedding significantly decreased in the vaccinated group when compared with the control ponies. All control ponies seroconverted after experimental infection with A/eq2/Sydney/2888-8/07 whereas only 1 out of 7 vaccinated ponies had a significant increase in antibody. Duvaxyn IE-T Plus therefore reduced clinical signs and virus shedding when ponies were challenged with A/eq2/Sydney/2888-8/07 (H3N8), 2 weeks after a second dose of vaccine.

Detecting autism spectrum disorder from early intervention charts: methodology and preliminary findings.

Federal laws mandating a "single point of entry" for early intervention (EI) create a potential database for surveillance of early childhood disabilities. This study evaluated EI records for estimating rates of autism spectrum disorder (ASD) using a chart abstraction protocol, with good interrater agreement (k = .86). Sampling 304 EI records yielded a point prevalence of (per 1,000) 8.5 (95% CI: 4.8-10.9) and a cumulative incidence of 7.4 (95% CI: 5.5-12.4). These rates are similar to recent published estimates. Additionally, the male-to-female ratio for autism, and rates of other developmental disorders were found to be consistent with current literature. These results suggest that local systems EI records may provide an excellent resource for ASD surveillance and research.

Both soluble and membrane-anchored forms of Felid herpesvirus 1 glycoprotein G function as a broad-spectrum chemokine-binding protein.

Recently, glycoprotein G (gG) of several alphaherpesviruses infecting large herbivores was shown to belong to a new family of chemokine-binding proteins (vCKBPs). In the present study, the function of Felid herpesvirus 1 (FeHV-1) gG as a vCKBP was investigated and the following conclusions were reached: (i) FeHV-1 secreted gG is a high-affinity broad-spectrum vCKBP that binds CC, CXC and C chemokines; (ii) gG is the only vCKBP expressed by FeHV-1 that binds CCL3 and CXCL1; (iii) secreted gG blocks chemokine activity by preventing their interaction with high-affinity cellular receptors; (iv) the membrane-anchored form of gG expressed on the surface of infected cells is also able to bind chemokines; and (v) the vCKBP activity is conserved among different field isolates of FeHV-1. Altogether, these data demonstrate that FeHV-1 gG is a new member of the vCKBP-4 family. Moreover, this study is the first to demonstrate that gG expressed at the surface of FeHV-1-infected cells can also bind chemokines.

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in b16 melanoma cells.

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Balloon cell melanoma in three dogs: a histopathological, immunohistochemical and ultrastructural study.

Balloon cell melanoma, a variant of malignant melanoma, has been reported on rare occasions in animals and is uncommon in man. Such tumours have variable numbers of large, round to polygonal cells with abundant, clear, often vacuolated cytoplasm containing fine melanin granules and variable amounts of lipid. This report describes balloon cell melanomas in three dogs. Immunohistochemically, these tumours showed reactions similar to those of human melanomas when tested with antibodies against S-100 protein, neuron-specific enolase (NSE) and vimentin. Electron microscopically, numerous heterogeneous melanosomes were demonstrated in the balloon cell cytoplasm of one tumour. Although balloon cell melanoma apparently occurs infrequently in dogs, it should always be considered in the differential diagnosis of neoplasms containing clear cells.

Genetic distances among North American, British, and West African house fly populations (Musca domestica L.).

Allozyme and mitochondrial gene diversities were estimated in house flies, Musca domestica L. (Diptera: Muscidae), sampled in Iowa, USA; Berkshire, England; and Kudang, The Gambia. Comparison of genomic allele frequencies among the three populations indicated small differences between the English and American samples but very large distances between English or American and the African. The FST statistic was 0.65 +/- 0.09 for allozymes. Pairwise FST was 0.14 between the English and the American samples; FST was 0.65 between the African population and the English and American. Mitochondrial variation in the same flies was assessed by SSCP methods which revealed nine haplotypes, none of which were shared in common. FST was 0.637 for the mitochondrial haplotypes. The research indicates greatly restricted gene flow between Africa and the temperate regions.

Cellular effects of leishmanial tubulin inhibitors on L. donovani.

To aid our investigation of tubulin as an antileishmanial drug target, the effects of the mammalian antimicrotubule agents ansamitocin P3, taxol, and hemiasterlin on Leishmania donovani promastigotes were described. These drugs affected the assembly of purified leishmanial tubulin and inhibited the growth of L. donovani promastigotes at micromolar concentrations. When promastigotes were treated with these agents, mitotic partitioning of nuclear DNA and cytokinesis were usually inhibited. The spatial orientation of kinetoplasts was often disturbed, suggesting a role for microtubules in the segregation of these organelles during mitosis. Aberrant cell types produced in drug-treated cultures included parasites with one nucleus and two geometrically distinct kinetoplasts, parasites with multiple kinetoplasts, and cytoplasts containing a kinetoplast but no nucleus. A subset of unique cell types, parasites containing two nuclei, a spindle fiber, and two geometrically distinct kinetoplasts, were observed in hemiasterlin-treated cultures. Flow cytometric analysis of L. donovani promastigotes treated with these three drugs indicated a dramatic shift toward the G2 + M phase of the cell cycle, with some cells containing four times the amount of DNA present in G1. These results were used to evaluate the cellular effects of WR85915, an aromatic thiocyanate with in vitro antileishmanial and anti-tubulin activity, on L. donovani. Treatment of parasites with WR85915 did not produce the unusual cell types described above and did not cause the accumulation of parasites in G2 + M, suggesting that WR85915 acts on target(s) in Leishmania in addition to tubulin. These studies validate tubulin as a suitable antileishmanial drug target and provide criteria to assess the cellular mechanism of action of new candidate antileishmanial agents.

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells.

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

A multimethod assessment of behavioral and emotional adjustment in children with asthma.

OBJECTIVE: Examine behavioral adjustment and emotion regulation among 6-year-old children with asthma and a group of healthy controls. METHOD: Subjects were 81 children with asthma and 22 healthy controls. Asthma and allergy statuses were confirmed by objective measures. Emotional and behavioral functioning were assessed through parent report, child interview, and child participation in an emotional regulation paradigm. RESULTS: Maternal report revealed more internalizing and total behavior problems for children with asthma compared to controls. Child interview and behavioral observations of emotion regulation yielded no differences between groups. Severity of asthma was related to increased emotional difficulties by clinician interview and observation but not by maternal report. CONCLUSIONS: Two groups of children with asthma who have psychological difficulties include those with increased anxiety and those with poor emotion regulation and more asthma symptoms. Different measures of child adjustment yield a complex picture of the behavioral difficulties associated with pediatric asthma.

Ectromelia, vaccinia and cowpox viruses encode secreted interleukin-18-binding proteins.

Interleukin-18 (IL-18) is a proinflammatory cytokine that plays a key role in the activation of natural killer and T helper 1 cell responses principally by inducing interferon-gamma (IFN-gamma). Human and mouse secreted IL-18-binding proteins (IL-18BPs) have recently been described which block IL-18 activity but have no sequence similarity to membrane IL-18 receptors. Several poxvirus genes encode proteins with sequence similarity to IL-18BPs. Here we show that vaccinia, ectromelia and cowpox viruses secrete from infected cells a soluble IL-18BP (vIL-18BP) that may modulate the host antiviral response. The ectromelia virus protein was found to block NF-kappaB activation and induction of IFN-gamma in response to IL-18. The highly attenuated vaccinia virus modified virus Ankara encodes IL-18-binding activity, and thus deletion of the vIL-18BP may improve further the safety and immunogenicity of this promising human vaccine candidate. We confirm that molluscum contagiosum virus, a molluscipoxvirus that produces small skin tumours in immunocompetent individuals and opportunistic infections in immunodeficient AIDS patients, also encodes a related, larger vIL-18BP (gene MC54L). This protein may contribute to the lack of inflammatory response characteristic of molluscum contagiosum virus lesions. The expression of vIL-18BPs by distinct poxvirus genera that cause local or general viral dissemination, or persistent or acute infections in the host, emphasizes the importance of IL-18 in response to viral infections.

VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment.

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

Gender differences in temporal lobe structures of patients with schizophrenia: a volumetric MRI study.

OBJECTIVE: The temporal lobe and associated structures have been previously implicated in the neuroanatomy of schizophrenia. This study was designed to assess the potential influence of gender on the morphology of temporal lobe structures, including the superior temporal gyrus and the amygdala/hippocampal complex, in patients with schizophrenia and to examine whether schizophrenic patients differ morphologically in these structures from comparison subjects. METHOD: Magnetic resonance imaging was used to measure the volume of temporal lobe structures, including the superior temporal gyrus, the amygdala/hippocampal complex, and the temporal lobe (excluding the volumes of the superior temporal gyrus and amygdala/hippocampal complex), and two comparison areas--the prefrontal cortex and caudate--in 36 male and 23 female patients with schizophrenia and 19 male and 18 female comparison subjects. RESULTS: There was a significant main effect of diagnosis in the superior temporal gyrus and the amygdala/hippocampal complex, with smaller volumes in patients than in comparison subjects. There was a significant gender-by-diagnosis-by-hemisphere interaction for temporal lobe volume. Temporal lobe volume on the left was significantly smaller in male patients than in male comparison subjects. Female patients and female comparison subjects demonstrated no significant difference in temporal lobe volume. There were no statistically significant gender interactions for the superior temporal gyrus, the amygdala/hippocampal complex, or the comparison regions. CONCLUSIONS: These findings suggest that there may be a unique interaction between gender and the pathophysiologic processes that lead to altered temporal lobe volume in patients with schizophrenia.

Retrograde traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment.

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid-containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.

Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: a VPS45-dependent intracellular route and a VPS45-independent, endocytic route.

The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment. Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology. Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment. This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways. Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole. Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway. In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway.

Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole.

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.