Adsorption and aggregation properties of norovirus GI and GII virus-like particles demonstrate differing responses to solution chemistry

The transport properties (adsorption and aggregation behavior) of virus-like particles (VLPs) of two strains of norovirus ("Norwalk" GI.1 and "Houston" GII.4) were studied in a variety of solution chemistries. GI.1 and GII.4 VLPs were found to be stable against aggregation at pH 4.0-8.0. At pH 9.0, GI.1 VLPs rapidly disintegrated. The attachment efficiencies (a) of GI.1 and GII.4 VLPs to silica increased with increasing ionic strength in NaCl solutions at pH 8.0. The attachment efficiency of GI.1 VLPs decreased as pH was increased above the isoelectric point (pH 5.0), whereas at and below the isoelectric point, the attachment efficiency was erratic. Ca(2+) and Mg(2+) dramatically increased the attachment efficiencies of GI.1 and GII.4 VLPs, which may be due to specific interactions with the VLP capsids. Bicarbonate decreased attachment efficiencies for both GI.1 and GII.4 VLPs, whereas phosphate decreased the attachment efficiency of GI.1, while increasing GII.4 attachment efficiency. The observed differences in GI.1 and GII.4 VLP attachment efficiencies in response to solution chemistry may be attributed to differential responses of the unique arrangement of exposed amino acid residues on the capsid surface of each VLP strain.

Identification of a new mumps virus lineage by nucleotide sequence analysis of the SH gene of ten different strains

The SH gene and its flanking sequences have been analysed for 10 strains of mumps virus (MuV) and compared to 5 others. A new lineage has been identified among UK isolates. Changes in the transcription pattern could not be correlated with differences in the sequences of the F-SH and SH-HN intergenic regions of the genome.

Temporal and geographical distribution of measles virus genotypes

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 co-circulating 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 Halle, Mantooth and Horta-Barbosa strains as well as some wild-type isolates from that period.

Clonal expansion of hypermutated measles virus in a SSPE brain

Nucleotide sequence analysis was carried out to study genes encoding the matrix (M) protein of measles virus (MV) from several regions of the brain of a case of subacute sclerosing panencephalitis. This analysis revealed the presence of MV with 'wild-type' sequences as well as variants which had undergone at least five biased hypermutation events (U to C and A to G in the positive strand sequences). Despite the presence of MV variants with genes encoding the intact matrix protein open reading frame, M protein could not be detected in any of the brain regions. The distribution of virus variants was studied by cDNA cloning and sequence analysis and by in situ hybridization. The hypermutated viruses appeared to expand clonally throughout the brain of patient B.

Mutation screening and genotype:phenotype correlation in familial hypercholesterolaemia

The aim of this study was to develop a mutation screening protocol for familial hypercholesterolaemia (FH) patients and to assess genotype/phenotype effects in terms of pre-treatment lipid profiles and presentation of tendon xanthomata (TX). A total of 158 families with clinical definitions of possible (120) or definite (38) FH were studied using a tiered screening protocol. Mutations were identified in 52 families, 44 families showing 23 different LDLR gene defects and eight families showing the common Apo B100 gene defect R3500Q. LDLR defects were detected in various regions of the gene with 56% in the LDL binding domain (exons 2-6) and 37% in the EGF precursor homology domain (exons 7-14). The most common mutations were D461N(7), C210X(5), 932delA(5), and C163Y(4). Frameshift mutations accounted for 20% with nonsense 13%, mis-sense 35%, splice 3%, Apo B 13% and 2% large deletion, 13% of clinically definite FH remained undefined. In conclusion, DNA based diagnosis is possible in 79% (30/38) of clinically definite FH families and of the 120 possible FH families at the start of the screening program, 18% (22/120) now have defined mutations. Overall 60 families from the original 158 meet the clinical and/or genetic criteria for definite FH. Tendon xanthomata were present in only 58% (30/52) of genetically defined FH families, thus limiting its use as a strict diagnostic criteria. Families with low density lipoprotein receptor (LDLR) defects present with higher total and LDL cholesterol levels and a higher incidence of TX than do those with the common Apo B variant, and frameshift mutations appear to have the most severe presentation. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Genotype/phenotype correlations in familial hypercholesterolaemia

It is now possible to identify the specific gene defect in the majority of patients with familial hypercholesterolaemia. A potential benefit of this knowledge, in addition to helping with family screens, is to be able to predict the future clinical course. In order to do this, detailed genotype/phenotype correlation studies are required.

Detection of avian influenza virus by fluorescent DNA barcode-based immunoassay with sensitivity comparable to PCR

In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms.

Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: Implications for respiratory virus-induced cough reflex sensitivity

ABSTRACT (250 words)
BACKGROUND: The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Up-regulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurones and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression.
METHODS: IMR32 neuroblastoma cells were differentiated in culture to express three TRP channels, TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus- induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1ß, IL-6 and IL-8 levels in response to infection.
RESULTS: Early up-regulation of TRPA1 and TRPV1 expression occurred 2 to4 hours post infection. This was independent of replicating virus as virus induced soluble factors alone were sufficient to increase channel expression 50 and 15 fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 hours (9.6 fold) and required virus replication rather than soluble factors
CONCLUSIONS We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes up-regulation of TRP channels by channel specific mechanisms. Increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.

Demonstration of antibodies in archival sera from Canadian seals reactive with a European isolate of phocine distemper virus

Sera from seals infected during the 1988 European epizootic of phocine distemper virus and sera from Canadian seals collected since 1972 have been tested for the presence of antibodies to morbillivirus. Approximately one third of the Canadian sera have been shown to contain anti-morbillivirus antibodies; the possibility that these populations of seals provided a source of infection for European seals is discussed.

Monospecific antibody to the haemagglutinin of measles virus

A hybrid between a murine myeloma cell line and spleen cells from a mouse immunized with measles has been produced. Two stable clones produce antibody with identical immunochemical and biological properties. This antibody reacts with the 76,000 mol. wt. protein present in the lysates and on the surface of cells persistently infected with measles. It exhibits HAI and neutralizing activity.

Neonatal Pain-Related Stress and NFKBIA Genotype Are Associated with Altered Cortisol Levels in Preterm Boys at School Age

Neonatal pain-related stress is associated with elevated salivary cortisol levels to age 18 months in children born very preterm, compared to full-term, suggesting early programming effects. Importantly, interactions between immune/inflammatory and neuroendocrine systems may underlie programming effects. We examined whether cortisol changes persist to school age, and if common genetic variants in the promoter region of the NFKBIA gene involved in regulation of immune and inflammatory responses, modify the association between early experience and later life stress as indexed by hair cortisol levels, which provide an integrated index of endogenous HPA axis activity. Cortisol was assayed in hair samples from 128 children (83 born preterm =32 weeks gestation and 45 born full-term) without major sensory, motor or cognitive impairments at age 7 years. We found that hair cortisol levels were lower in preterm compared to term-born children. Downregulation of the HPA axis in preterm children without major impairment, seen years after neonatal stress terminated, suggests persistent alteration of stress system programming. Importantly, the etiology was gender-specific such that in preterm boys but not girls, specifically those with the minor allele for NFKBIA rs2233409, lower hair cortisol was associated with greater neonatal pain (number of skin-breaking procedures from birth to term), independent of medical confounders. Moreover, the minor allele (CT or TT) of NFKBIA rs2233409 was associated with higher secretion of inflammatory cytokines, supporting the hypothesis that neonatal pain-related stress may act as a proinflammatory stimulus that induces long-term immune cell activation. These findings are the first evidence that a long-term association between early pain-related stress and cortisol may be mediated by a genetic variants that regulate the activity of NF-?B, suggesting possible involvement of stress/inflammatory mechanisms in HPA programming in boys born very preterm. © 2013 Grunau et al.

A Longitudinal Study of Stargardt Disease: Quantitative Assessment of Fundus Autofluorescence, Progression and Genotype Correlations

PURPOSE To characterise subtypes of fundus autofluorescence (AF), the progression of retinal atrophy and correlate these findings with genotype in Stargardt Disease. METHODS Full clinical examination and AF imaging was undertaken in 68 patients with Stargardt Disease. The baseline data were compared with those at follow-up. Patients were classified into three AF subtypes: type 1 had a localised low signal at the fovea surrounded by a homogeneous background; type 2 had a localised low signal at the macula surrounded by a heterogeneous background with numerous foci of abnormal signal; type 3 had multiple low signal areas at the posterior pole with a heterogeneous background. At baseline, there were 19 patients with type 1, 41 with type 2, and 8 with type 3. The areas of reduced AF signal were measured and rate of atrophy enlargement (RAE) was calculated as the difference of the atrophy size over time (mm2) divided by the follow-up interval (yrs). Molecular screening of ABCA4 was undertaken. RESULTS The mean follow-up interval was 9.1 years. 42% of type 1 progressed to type 2, and 12% of type 2 progressed to type 3. RAE (mm2/yr) based upon baseline AF subtypes was significantly different; 0.06 in type 1, 0.67 in type 2, and 4.37 in type 3. ABCA4 variants were identified in 57 patients. There was a significant association between AF subtype and genotype. CONCLUSIONS The AF pattern at baseline influences the enlargement of atrophy over time and has genetic correlates. These data are likely to assist in the provision of counselling on prognosis in Stargardt Disease and be valuable for future clinical trials.

Squirrelpox Virus: Assessing Prevalence, Transmission and Environmental Degradation

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25°C) and dry conditions than in cooler (5 and 15°C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species.

Respiratory syncytial virus infections enhance cigarette smoke induced COPD in mice

Respiratory syncytial viral (RSV) infections are a frequent cause of chronic obstructive pulmonary disease (COPD) exacerbations, which are a major factor in disease progression and mortality. RSV is able to evade antiviral defenses to persist in the lungs of COPD patients. Though RSV infection has been identified in COPD, its contribution to cigarette smoke-induced airway inflammation and lung tissue destruction has not been established. Here we examine the long-term effects of cigarette smoke exposure, in combination with monthly RSV infections, on pulmonary inflammation, protease production and remodeling in mice. RSV exposures enhanced the influx of macrophages, neutrophils and lymphocytes to the airways of cigarette smoke exposed C57BL/6J mice. This infiltration of cells was most pronounced around the vasculature and bronchial airways. By itself, RSV caused significant airspace enlargement and fibrosis in mice and these effects were accentuated with concomitant smoke exposure. Combined stimulation with both smoke and RSV synergistically induced cytokine (IL-1a, IL-17, IFN-c, KC, IL-13, CXCL9, RANTES, MIF and GM-CSF) and protease (MMP-2, -8, -12, -13, -16 and cathepsins E, S, W and Z) expression. In addition, RSV exposure caused marked apoptosis within the airways of infected mice, which was augmented by cigarette smoke exposure. RSV and smoke exposure also reduced protein phosphatase 2A (PP2A) and protein tyrosine phosphates (PTP1B) expression and activity. This is significant as these phosphatases counter smoke-induced inflammation and protease expression. Together, these findings show for the first time that recurrent RSV infection markedly enhances inflammation, apoptosis and tissue destruction in smoke-exposed mice. Indeed, these results indicate that preventing RSV transmission and infection has the potential to significantly impact on COPD severity and progression.

Efficacy of Tenofovir 1% Vaginal Gel in Reducing the Risk of HIV-1 and HSV-2 Infection

Human Immunodeficiency Virus (HIV) is a retrovirus that can result in rare opportunistic infections occurring in humans. The onset of these infections is known as Acquired Immune Deficiency Syndrome (AIDS). Sexual transmission is responsible for the majority of infections 1, resulting in transmission of HIV due to infected semen or vaginal and cervical secretions containing infected lymphocytes. HIV microbicides are formulations of chemical or biological agents that can be applied to the vagina or rectum with the intention of reducing the acquisition of HIV. Tenofovir is an NRTI that is phosphorylated by adenylate kinase to tenofovir diphosphate, which in turn competes with deoxyadeosine 5’-triphosphate for incorporation into newly synthesized HIV DNA. Once incorporated, tenofovir diphosphate results in chain termination, thus inhibiting viral replication. Tenofovir has been formulated into a range of vaginal formulations, such as rings, tablets gels and films. It has been shown to safe and effective in numerous animal models, while demonstrating safety and acceptability in numerous human trials. The most encouraging results came from the CAPRISA 004 clinical trial which demonstrated that a 1% Tenofovir vaginal gel reduced HIV infection by approximately 39%.