Comparative analysis of the complete genome sequences of two Australian origin live attenuated vaccines of infectious laryngotracheitis virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in poultry. Live attenuated ILTV vaccines have been used extensively to help control outbreaks of disease. Two Australian-origin attenuated vaccine strains, SA2 and A20 ILTV, are commercially available and are in frequent use in Australia. Both these vaccines are of chicken embryo origin (CEO). The A20 ILTV strain was developed from the SA2 ILTV strain by sequential passage of SA2 ILTV in tissue culture in order to reduce its residual virulence. Previous studies in our laboratories have demonstrated the greater attenuation of A20 ILTV under controlled experimental conditions, but the genetic basis of the in vivo phenotypes of A20 and SA2 ILTV has not been elucidated. In this study, the genetic differences between A20 and SA2 ILTV were examined by performing complete genome sequencing and comparative analysis. The genome sequences were also compared to a reference sequence from another CEO ILTV vaccine (Serva ILTV: GenBank accession number HQ_630064) of European-origin. Additional in ovo studies to assess cell to cell spread were performed in order to allow further comparisons of the pathogenicity of SA2 and A20 ILTV. The sequencing results showed that the genome sizes of SA2 and A20 ILTV were 152,975 and 152,978 bp, respectively, while Serva ILTV had a genome size of 152,630 bp. The genomes of SA2 and A20 ILTV shared 99.9% nucleotide sequence identity with each other, but only 99.2% identity with Serva ILTV. In complete genome alignments between SA2 and A20 ILTV, a total of 24 single nucleotide polymorphisms (SNPs) were identified, but only two of these were non-synonymous. These were located in the ORF B and UL15 genes. Four indels were detected in non-coding regions. The findings from this study demonstrate the general genetic stability of ILTV, but also show that non-synonymous changes in the ORF B and UL15 genes have arisen following tissue culture passage of SA2 ILTV to produce the A20 vaccine. It is likely that these non-synonymous changes are related to the greater attenuation of A20 ILTV compared to SA2 ILTV, and to the reduced ability of A20 ILTV to spread from cell to cell, as observed in this study. The results from this study also demonstrate the divergence between the genomes of the Australian-origin ILTV vaccine strains and the Serva vaccine strain.     

Public Health Nurses and the delivery of quality nursing care in the community

Aim. The aim of the study was to explore factors which impact on quality nursing care in the community from the Public Health Nurse's (PHN) perspective. Background. Public Health Nursing has significantly evolved over the past few years with the delivery of quality nursing being a focus point. This study explores factors that impact upon the delivery of quality care in Public Health Nursing in Ireland. The findings provide an opportunity for an additional perspective to be included in the existing international findings and act as a starting point from which further research can be built. Method. A qualitative method using semi‐structured interviews were conducted. Interviews were taped and content analysed. Findings. Four main categories emefrged from the data, namely role change, components of quality nursing care, barriers to quality nursing care and the factors that facilitate the delivery of quality nursing care in the community. PHNs strive for evidence‐based practice; they acknowledged their inability to achieve this and referred to factors that inhibited them from reaching their goal. Conclusion. Enhanced education for PHNs will equip them in the delivery of a quality service and have a positive impact on patient care. Better communication is required between PHNs, line managers and the multidisciplinary team. The delivery of community services need to be reviewed and developed further in accordance with the health strategy policy. Relevance to clinical practice. This study has identified the evolution in clinical practice associated with the changing role and scope of Public Health Nursing. Clinical practice has evolved over time to incorporate societal change, technological advances and the delivery of an evidence‐based service responsive to identified need. This study identified the presence of an increase in the specialist clinical work being undertaken as a result of new technological advances entering the community working environment.     

Salvage versus amputation: Utility of mangled extremity severity score in severely injured lower limbs

Background:

The purpose of the present study was to evaluate the clinical utility of Mangled extremity severity score (MESS) in severely injured lower limbs.

Materials and Methods:

Retrospectively 25 and prospectively 36 lower limbs in 58 patients with high-energy injuries were evaluated with the use of MESS, to assist in the decision-making process for the care of patients with such injuries. Difference between the mean MESS scores for amputated and salvaged limbs was analyzed.

Results:

In the retrospective study 4.65 (4.65 ± 1.32) was the mean score for the salvaged limbs and 8.80 (8.8 ± 1.4) for the amputated limbs. In the prospective study 4.53 (4.53 ± 2.44) was the mean score for the salvaged limbs and 8.83 (8.83 ± 2.34) for the amputated limbs. There was a significant difference in the mean scores for salvaged and amputated limbs. Retrospective 21 (84%) and prospective 29 (80.5%) limbs remained in the salvage pathway six months after the injury.

Conclusion:

MESS could predict amputation of severely injured lower limbs, having score of equal or more than 7 with 91% sensitivity and 98% specificity. There was a significant difference in the mean MESS scores in the prospective study (n=36), 4.53 (4.53 ± 2.44) in thirty salvaged limbs (83.33%) and 8.83 (8.83 ± 2.34) in six amputated limbs (16.66%) with a P-value 0.002 (P-value < 0.01). Similarly there was a significant difference in the mean MESS score in the retrospective study (n=25), 4.65 (4.65 ± 1.32) in twenty salvaged limbs (80%) and 8.80 (8.8 ± 1.4) in five amputated limbs (20%) with a P-value 0.00005 (P-value < 0.01). MESS is a simple and relatively easy and readily available scoring system which can help the surgeon to decide the fate of the lower extremity with a high-energy injury.     

Identification of autosomal recessive disease loci using out-bred nuclear families

Autozygosity mapping has been a powerful method for the identification of autosomal recessive disease genes. However, the approach is limited by the availability of suitable consanguineous pedigrees. While rare autosomal recessive diseases are overrepresented in consanguineous families, a significant proportion of affected patients nonetheless originate in families where the parents are apparently unrelated. However, due to their relative rarity and the heterogeneity of disease alleles, it has proved difficult to use these patients to identify disease loci. Therefore, we developed "Phaser," a computer application that is able to infer the phase of SNP alleles and so haplotype entire chromosomes in small nuclear families (http://dna.leeds.ac.uk/Phaser). Once the index case's chromosomes have been haplotyped, it is then possible to deduce those of the parents and subsequently identify the parental origin of all the siblings' DNA. By combining information from a small number of nuclear families, it may then be possible to identify linkage to the recessive disease locus, in both in-bred and out-bred families. We have illustrated the program's utility by using it to correctly identify both the cystic fibrosis locus (using two unrelated compound heterozygous CEPH families) and a new gene mutated in early-onset myopathy with respiratory distress and dysphagia locus in a single consanguineous pedigree.     

Genetic diagnosis of familial breast cancer using clonal sequencing

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at‐risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA‐deficient breast cancers. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Brain death in infants: evaluation with Doppler US

In a preliminary study, nine infants with a clinically determined diagnosis of brain death were examined with duplex pulsed Doppler ultrasonography (US) through the anterior fontanelle. Flow velocity measurements were made in the intracranial internal carotid artery and anterior cerebral artery. Resistive index (RI) was calculated in each patient and used as an indicator of diastolic flow. Eight of nine infants showed markedly elevated RI (100%-191%) with reversal of diastolic flow. One infant had low RI (42%-58%) with preserved systolic and diastolic flow until death. The authors believe that the elevation of RI with diastolic flow reversal seen in these patients is a reflection of increased intracranial pressure and is a sign of poor prognosis when present on serial examinations. Cranial duplex pulsed Doppler US is a useful, noninvasive tool in the diagnosis of brain death in infants but must be carefully correlated with clinical examination and other diagnostic tests.     

Lentivector Transduction Improves Outcomes Over Transplantation of Human HSCs Alone in NOD/SCID/Fabry Mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A) activity that results in progressive globotriaosylceramide (Gb₃) deposition. We created a fully congenic nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/Fabry murine line to facilitate the in vivo assessment of human cell-directed therapies for Fabry disease. This pure line was generated after 11 generations of backcrosses and was found, as expected, to have a reduced immune compartment and background α-gal A activity. Next, we transplanted normal human CD34⁺ cells transduced with a control (lentiviral vector-enhanced green fluorescent protein (LV-eGFP)) or a therapeutic bicistronic LV (LV-α-gal A/internal ribosome entry site (IRES)/hCD25). While both experimental groups showed similar engraftment levels, only the therapeutic group displayed a significant increase in plasma α-gal A activity. Gb₃ quantification at 12 weeks revealed metabolic correction in the spleen, lung, and liver for both groups. Importantly, only in the therapeutically-transduced cohort was a significant Gb₃ reduction found in the heart and kidney, key target organs for the amelioration of Fabry disease in humans.