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Canine distemper virus (CDV) and phocine distemper (PDV) are closely-related members of the Paramyxoviridae family, genus morbillivirus, in the order Mononegavirales. CDV has a broad host range among carnivores. PDV is thought to be derived from CDV through contact between terrestrial carnivores and seals. PDV has caused extensive mortality in Atlantic seals and other marine mammals, and more recently has spread to the North Pacific Ocean. CDV also infects marine carnivores, and there is evidence of morbillivirus infection of seals and other species in Antarctica. Recently, CDV has spread to felines and other wildlife species in the Serengeti and South Africa. Some CDV vaccines may also have caused wildlife disease. Changes in the virus haemagglutinin (H) protein, particularly the signaling lymphocyte activation molecule (SLAM) receptor binding site, correlate with adaptation to non-canine hosts. Differences in the phosphoprotein (P) gene sequences between disease and non-disease causing CDV strains may relate to pathogenicity in domestic dogs and wildlife. Of most concern are reports of CDV infection and disease in non-human primates raising the possibility of zoonosis. In this article we review the global occurrence of CDV and PDV, and present both historical and genetic information relating to these viruses crossing species barriers.

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In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.

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BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling. RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension. CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

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AIMS: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry. METHODS: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real-time reverse transcriptase polymerase chain reaction. RESULTS: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections. CONCLUSIONS: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.

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Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has now been described globally, as a clinically significant pathogen, particularly associated with skin and soft tissue infections, including abscesses, cellulitis and furunculosis. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of healthcare-associated HA-MRSA-related wound infection, as well as honey's ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. Although previous studies have examined the antimicrobial activity of honey against HA-MRSA, such data are limited regarding the activity of honey against this emerging type of MRSA. CA-MRSA (n=6 isolates), was examined for its susceptibility to natural honey (n=3 honey produced from bees in Northern Ireland and one commercial French honey). Results demonstrated that all honey was able to reduce the cultural count of all CA-MRSA from approximately 10(6) colony-forming units (cfus) (mean = 6.46 log10 cfu/g) to none detectable within 24h of co-culture of separate CA-MRSA organisms individually with all four-honey types examined. Subsequent non-selective enrichment of honey demonstrated that inoculated honey remained positive for CA-MRSA until 72h postinoculation, after which point no culturable organisms could be detected. This study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate if this antimicrobial activity has any clinical application.

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A proportion of Hodgkin lymphoma (HL) cases are causally associated with the Epstein-Barr virus (EBV) but the aetiology of the remaining cases remains obscure. Over the last 3 decades several studies have found an association between HL and measles virus (MV) including a recent cohort study describing the detection of MV antigens in Hodgkin and Reed-Sternberg cells, the tumour cells in HL. In the present study we looked at the relationship between history of MV infection and risk of developing HL in a population-based, case/control study of HL. In addition we used immunohistochemistry and RT-PCR to look for direct evidence of MV in HL biopsies. There was no significant difference in the proportion of cases reporting previous measles compared to controls in the entire data set or when young adults were considered separately. Using a robust immunohistochemical assay for MV infection, we failed to find evidence of MV in biopsies from 97 cases of HL and RT-PCR studies similarly gave negative results. This study therefore provides no evidence that MV is directly involved in the development of HL. However, when age at first reported MV infection was investigated, significant differences emerged with children infected before school-age having higher risk, especially of EBV-ve HL, when compared with children infected at older ages; the interpretation of these latter results is unclear.

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Phenotypic speciation of foodborne Bacillus spp. remains problematic in terms of obtaining a reliable identification. In this study, we wished to identify several bacterial isolates from honey produced in Northern Ireland, and which belonged to the genus Bacillus, through employment of a molecular identification scheme based on PCR amplification of universal regions of the 16S rRNA operon in combination with direct automated sequencing of the resulting amplicons. Seven samples of honey and related materials (propolis) were examined microbiologically and were demonstrated to have total viable counts (TVC) ranging from <100 to 1700 colony-forming units/g. No yeasts or filamentous fungi were isolated from the honey materials. Several bacterial isolates were identified using this method, yielding two different genera (Paenibacillus and Bacillus), as well as four Bacillus species, namely Bacillus pumilus, B. licheniformis, B. subtilis and B. fusiformis, with B. pumilus the most frequently identified species present. When the use of molecular identification methods is justified, employment of partial 16S rDNA PCR and sequencing provides a valuable and reliable method of identification of Bacillus spp. from foodstuffs and negates associated problems of conventional laboratory and phenotypic identification.

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The gene encoding the large (L) protein and the genome termini of the dolphin strain of cetacean morbillivirus (CeMV) were sequenced. The CeMV genome is 15702 nucleotides long and has been compared with other available morbillivirus genome sequences in regards to the "rule of six" and the "phase" of any particular nucleotide, defined as its position within a given hexamer, which here is defined as a group of six nucleotides starting from the 3' end of the genomic RNA. With exception of the position of the start of the F gene, the phase of the transcription start sites of each gene is strictly conserved between the morbilliviruses, but each gene is in a different phase. The lengths of gene transcripts differ between viruses by multiples of six nucleotides with exception of the M and F transcripts. The differences between the various morbilliviruses result from deletions or insertions of multiples of six nucleotides in the 3' and 5' UTRs of the different viral genes. The four bases were distributed non-randomly over the six positions in the hexamer boxes. However, the distribution patterns of each of the four bases indicated that multiples of three were more prevalent than those of six nucleotides. This reflected the positions of nucleotides in codons and codon usage in the reading frames. The L protein of CeMV was found to be 2183 amino acids in length and similar to that of MV and RPV. The CeMV L protein sequence was found to be equidistant between those of the CDV/PDV and MV/RPV subgroups of the morbilliviruses. This concurs with the analyses carried out on the other structural proteins.

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Epidemiological reports describe a strong association between prenatal human influenza viral infection and later development of schizophrenia. Postmodern human brain studies, however, indicate a lack of gliosis in schizophrenic brains presumably secondary to absence of glial cells during the second trimester viral infection in utero. We hypothesized that human influenza infection in day 9 pregnant mice would alter the expression of glial fibrillary acidic protein (GFAP, an important marker of gliosis, neuron migration, and reactive injury) in developing brains of postnatal days 0, 14 and 35 mice. Determination of cellular GFAP immunoreactivity (IR) expressed as cell density in cortex and hippocampus of control and experimental brains showed increases in GFAP-positive density in exposed cortical (P = 0.03 day 14 vs control) and hippocampal cells (P = 0.035 day 14, P = 0.034 day 35). Similarly, ependymal cell layer GFAP-IR cell counts showed increases with increasing brain age from day 0, to days 14 and 35 in infected groups (P = 0.037, day 14) vs controls. The GFAP-positive cells in prenatally exposed brains showed 'hypertrophy' and more stellate morphology. These results implicate a significant role of prenatal human influenza viral infection on subsequent gliosis, which persists throughout brain development in mice from birth to adolescence.

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SNAP-25 levels were measured in ventral hippocampus in subjects with unipolar depression (n = 12), bipolar disorder (n = 13), schizophrenia (n = 15) and controls (n = 15) using quantitative immunocytochemistry. SNAP-25 levels were reduced significantly in stratum oriens of bipolar patients compared with controls (p < 0.05); they were also reduced significantly in st. oriens (p < 0.01 vs schizophrenia), in alveous (p < 0.01 vs schizophrenia) and in presubiculum (p < 0.05 vs depressed). SNAP-25 levels were also reduced in several layers of schizophrenics, only significantly so in st. granulosum (p < 0.05 vs controls). In contrast, depressed SNAP-25 levels increased in st. moleculare (p < 0.01 vs schizophrenics) and presubiculum (p < 0.05 vs controls and bipolars; p < 0.01 vs schizophrenics). SNAP-25 values were not affected by age, sex, race, post-mortem interval, brain pH, side of brain, age of onset of disease, family history of psychiatric disease, drug or alcohol use, antipsychotic drug treatment, or mode of death. The reported changes in SNAP-25 levels appear to be disease specific, separating synaptic pathology in unipolar depression from that observed in schizophrenia and bipolar disorders.

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Accumulation of neurobiological knowledge points to neurodevelopmental origins for certain psychotic and mood disorders. Recent landmark postmortem reports implicate Reelin, a secretory glycoprotein responsible for normal lamination of brain, in the pathology of schizophrenia and bipolar disorders. We employed quantitative immunocytochemistry to measure levels of Reelin protein in various compartments of hippocampal formation in subjects diagnosed with schizophrenia, bipolar disorder and major depression compared to normal controls. Significant reductions were observed in Reelin-positive adjusted cell densities in the dentate molecular layer (ANOVA, P < 0.001), CA4 area (ANOVA, P < 0.001), total hippocampal area (ANOVA, P < 0.038) and in Reelin-positive cell counts in CA4 (ANOVA, P < 0.042) of schizophrenics vs controls. Adjusted Reelin-positive cell densities were also reduced in CA4 areas of subjects with bipolar disorder (ANOVA, P < 0.001) and nonsignificantly in those with major depression. CA4 areas were also significantly reduced in schizophrenic (ANOVA, P < 0.009) patients. No significant effects of confounding variables were found. The exception was that family history of psychiatric illness correlated strongly with Reelin reductions in several areas of hippocampus (CA4, adjusted cell density, F = 13.77, P = 0.001). We present new immunocytochemical evidence showing reductions in Reelin expression in hippocampus of subjects with schizophrenia, bipolar disorder and major depression and confirm recent reports documenting a similar deficit involving Reelin expression in brains of subjects with schizophrenia and bipolar disorder.

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This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert FAN aerobic blood culture material examined.

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Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

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We have developed a simple, rapid and reliable method for specifically amplifying and cloning full-length HLA-B genes from genomic DNA. Using this methodology we characterized three alleles of interest at the molecular level. Two of the alleles appeared in our routine class I PCR-SSOP typing system, a variant of B*5801 found in the Daudi cell line and RCE 56 and a variant of B*4101 found in a number of volunteer donors on our Bone Marrow Donor Registry. The third, a variant B35 allele found in RCE 80, was first identified as unusual by serology. Our sequencing analysis of exon 2 and exon 3 identified two of these alleles as the recently reported novel HLA-B*5802 and HLA-B*4102 alleles, while the third represents a new B35 allele officially designated B*3513.

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Sequence data for the nucleocapsid protein (N) gene of the porpoise morbillivirus including the very conserved middle section of the protein and the hypervariable C terminus are reported. Analysis of dissimilarity indices based on an alignment of the N proteins of various morbilliviruses identifies a variable region of the N protein from amino acids residues 121 to 145 and a hypervariable part from amino acids 400 to 517. This type of analysis can be usefully applied when protein sequences of five or more morbillivirus species are available. Regions of variability between species identified by this index also represent regions of variation within one species e.g. measles virus (MV). Hence, comparative analysis of different morbilliviruses provides an insight into the potentially variable parts of viral proteins. From the great and unexplained nucleotide sequence conservation observed within MV, it would appear that the various morbilliviruses have diverged from each other a very long time ago. However, the data do not yet allow us to estimate the time span of these divergences. The relatedness and the number of different morbillivirus species provides a unique database for study of the evolution of RNA viruses.

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The nucleotide sequence encoding the C terminus of the nucleocapsid protein of measles virus (MV) is the most variable in the genome. The sequence of this region is reported for 21 new MV strains and for virus RNA obtained from cases of subacute panencephalitis (SSPE) tissue. The nucleotide sequence of a total of 65 MV strains has been analysed using the CLUSTAL program to determine the relationships between the strains. An unrooted tree shows that eight different genotypes can be discerned amongst the sequences analysed so far. The data show that the C-terminal coding sequence of the nucleocapsid gene, although highly variable between strains, is stable in a given strain and does not appear to diverge in tissue culture. It therefore provides a good 'signature' sequence for specific genotypes. The sequence of this region can be used to discriminate new imported viruses from old 'endemic' strains of MV in a geographical area. The different genotypes are not geographically restricted although some appear to be the mainly 'endemic' types in large areas of the world. In global terms there appears to be at least four cocirculating genotypes of MV. The low level of divergence in the Edmonston lineage group isolated before 1970 indicates that some isolates are probably laboratory contaminants. This applies to some SSPE isolates such as the Hallé, Mantooth and Horta-Barbosa strains as well as some wild-type isolates from that period.

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Morbilliviruses have been isolated from stranded dolphins and porpoises. The present paper describes the cloning and sequencing of the porpoise morbillivirus (PMV) F gene and of the dolphin morbillivirus (DMV) M and F genes and their flanking regions. The gene order of the DMV genome appeared to be identical to that of other morbilliviruses. A genomic untranslated region of 837 nucleotides was found between the translated DMV M and F gene regions. The predicted DMV M protein were highly conserved with those of other morbilliviruses. Both the deduced PMV and DMV F0 proteins exhibited three major hydrophobic regions as well as a cysteine rich region, a leucine zipper motif and a cleavage motif allowing cleavage of the F0 protein into F1 and F2 subunits. Apparently the DMV F0 cleavage motif was not modified by adaptation of DMV to Vero cells. The predicted PMV and DMV F proteins were 94% identical. Comparisons with the corresponding sequences of other morbilliviruses demonstrated that the cetacean morbillivirus does not derive from any known morbillivirus but represents an independent morbillivirus lineage.

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