Characterisation of the H5 and N1 genes of an Indonesian highly pathogenic Avian Influenza virus isolate by sequencing of multiple clone approach

Hemagglutinin and neuraminidase are the main antigenic determinants of highly pathogenic avian influenza (HPAI) virus. The features of these surface glycoproteins have been intensively studied at the molecular level. The objective of this research was to characterise the genes encoding these glycoproteins by sequencing of multiple clones. The H5 and N1 genes of isolate A/duck/Tangerang/Bbalitvet-ACIAR-TE11/2007 were each amplified in one or two fragments using reverse transcriptase-PCR (RT-PCR), and subsequently cloned into pGEM-T Easy TA cloning system. The sequencing result demonstrated high homology between respective clones but with several variations that were identified as single nucleotide polymorphisms (SNPs). A total of 1,707 base pair and 1,350 base pair of H5 and N1 genes respectively were successfully assembled from multiple clones containing the genes of interest. The features of both H5 and N1 genes from this isolate resemble the typical characteristics of Indonesian strains of H5N1 virus from sub-clade 2.1.3. Key Words: Avian Influenza, Characterization, Gene Cloning, Hemagglutinin, Neuraminidas

'Just Ask Me': The Importance of Respectful Relationships Within Schools

An inclusive approach to education requires schools and educators to address the support needs and individual predispositions of all students. Our research highlights the crucial importance of effective and respectful communication with autistic students to facilitate their successful participation in schools. This paper explores the experiences of 24 autistic individuals aged 16–67 years, through synchronous semi-structured interviews and written responses. The research team comprised both autistic and allistic (non-autistic) researchers, who worked together to design the overall project, collect interview data, and analyse the data. Relationships were frequently discussed by participants and the importance of positive relationships was positioned as key to successful participation within educational contexts. Particularly damaging were assumptions made by teachers concerning individual ability based on labels given. Participants recalled ongoing challenges with resisting stereotypes and managing stigma, while trying to craft a positive autistic identity and advocate for rightful supports for their education. At the core of these negotiations were positive relationships, and teachers who asked participants what their needs were, and then listened and proactively responded to their answers. Recommendations for more positive schooling engagements with autistic young people are provided

“Peas in a pod”: Oral History Reflections on Autistic Identity in Family and Community by Late-Diagnosed Adults

In this paper, we report on a participatory oral history study documenting the lives of late-diagnosed autistic adults in Australia. We interviewed 26 autistic adults about their life history and the impact of late diagnosis. All were diagnosed after the age of 35, growing up in an era when autism was not well known. Using reflexive thematic analysis, we uncovered a rich body of reflections on shared Autistic identity and identified three major themes within that data set: 'conceptualising the Autistic family', 'creating Autistic community', and 'contesting Autistic identity'. Overall, the study provides insights into the active creation of shared Autistic identity and the importance of Autistic community to these late-diagnosed autistic adults

Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses

Large Scale Comparison of Innate Responses to Viral and Bacterial Pathogens in Mouse and Macaque

Viral and bacterial infections of the lower respiratory tract are major causes of morbidity and mortality worldwide. Alveolar macrophages line the alveolar spaces and are the first cells of the immune system to respond to invading pathogens. To determine the similarities and differences between the responses of mice and macaques to invading pathogens we profiled alveolar macrophages from these species following infection with two viral (PR8 and Fuj/02 influenza A) and two bacterial (Mycobacterium tuberculosis and Francisella tularensis Schu S4) pathogens. Cells were collected at 6 time points following each infection and expression profiles were compared across and between species. Our analyses identified a core set of genes, activated in both species and across all pathogens that were predominantly part of the interferon response pathway. In addition, we identified similarities across species in the way innate immune cells respond to lethal versus non-lethal pathogens. On the other hand we also found several species and pathogen specific response patterns. These results provide new insights into mechanisms by which the innate immune system responds to, and interacts with, invading pathogens

Characterisation of the H5 and N1 genes of an Indonesian highly pathogenic avian influenza virus isolate by sequencing of multiple clone approach

Hemagglutinin and neuraminidase are the main antigenic determinants of highly pathogenic avian influenza (HPAI) virus. The features of these surface glycoproteins have been intensively studied at the molecular level. The objective of this research was to characterise the genes encoding these glycoproteins by sequencing of multiple clones. The H5 and N1 genes of isolate A/duck/Tangerang/Bbalitvet-ACIAR-TE11/2007 were each amplified in one or two fragments using reverse transcriptase-PCR (RT-PCR), and subsequently cloned into pGEM-T Easy TA cloning system. The sequencing result demonstrated high homology between respective clones but with several variations that were identified as single nucleotide polymorphisms (SNPs). A total of 1,707 base pair and 1,350 base pair of H5 and N1 genes respectively were successfully assembled from multiple clones containing the genes of interest. The features of both H5 and N1 genes from this isolate resemble the typical characteristics of Indonesian strains of H5N1 virus from sub-clade 2.1.3

The decision-making processes of early childhood teachers when working with children experiencing parental separation and divorce

In this study, the pedagogical decision-making processes of 21 Australian early childhood teachers working with children experiencing parental separation and divorce were examined. Transcripts from interviews and a focus group with teachers were analysed using grounded theory methodology. The findings showed that as teachers interacted with young children experiencing parental separation and divorce, they reported using strategic, reflexive pedagogical decision-making processes. These processes comprised five stages: - (1) teachers constructing their knowledge; - (2) teachers thinking about their knowledge; - (3) teachers using decision-making schemas; - (4) teachers taking action, and; - (5) teachers monitoring action and evaluating. This understanding of teachers’ reflexive pedagogical decision-making is useful for identifying how teachers and educational leaders can support children experiencing parental separation and divorce or other life challenges

Teachers facilitating support for young children experiencing parental separation and divorce

This paper reports on a study of Australian early childhood teachers’ pedagogical practices with young children experiencing parental separation and divorce. Twenty-one semi-structured interviews and a focus group were conducted to explore the actions of teachers to support young children experiencing parental separation and divorce. A grounded theory approach was used to analyse data. Teachers reported actions that were focussed on constructing emotional, behavioural, and academic support for young children, as well as forming partnerships with parents, school personnel, and community members to assist. Results are discussed in terms of the implications for professional practice

Identification of non-essential loci within the Meleagrid herpesvirus 1 genome

Background: Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome. Methods: Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts. Results: Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture. Conclusions: The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome