Veterinary Science Collected Works - Theses

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Start date: 2014 × Subject: Avian viral pathogens × Subject: Infectious laryngotracheitis virus ×

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    Novel platforms for full genomic characterisation of avian pathogens directly from clinical samples
    Asif, Kinza ( 2021)
    Identification of the microbial strains involved in infectious disease is imperative for epidemiological investigation and to implement control strategies using vaccination. The conventional techniques used for strain identification including microneutralisation assays and polymerase chain reaction (PCR)-based assays such as PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-High resolution melt (PCR-HRM) curve analysis are not only cumbersome but also their results are difficult to interpret. Analysis of the whole genomes of the pathogens is considered the gold standard for the characterisation of strains. However, whole-genome sequencing (WGS) requires the in vitro isolation of the pathogens which can potentially introduce variations in the genome. In addition, some pathogens are not cultivable in vitro. Therefore, WGS directly from clinical tissues would be the most suitable option. In this study, four viral pathogens namely Fowl adenovirus (FAdV), avian hepatitis E virus (aHEV), fowlpox virus (FPV), and infectious laryngotracheitis virus (ITLV) affecting the hepatic, cutaneous, and respiratory tissues were targeted in four independent studies to assess the suitability of WGS directly from clinical tissues using Illumina and Nanopore sequencing platforms. To extract sequencing grade viral DNA/RNA directly from hepatic tissues, the liver homogenate was treated with 5% kaolin hydrated aluminium silicate to remove excess lipid tissue present in the liver before proceeding to the phenol-chloroform extraction. FAdV DNA extracted from treated tissue, resulted in the complete genome assembly of FAdV using both Illumina and Nanopore sequencing platforms. A similar extraction technique was used to extract aHEV RNA directly from liver tissues followed by long range RT-PCR and sequencing. . Analysis of the resultant WGS from clinical samples revealed that Australian aHEV isolates had emerged as a result of recombination between the US and European strains possibly following the importation of poultry into Australia and dissemination through vertical transmission. To evaluate the suitability of WGS directly from cutaneous tissues affected with FPV, homogenate of the affected comb tissue was subjected to DNA extraction and sequencing. The sequencing results were compared with the same FPV isolate grown in chorioallantoic membranes (CAMs). Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar, but not identical to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of SNPs in the two sequences when compared to the reference genome, essentially classifying the two sequences as the same strain, but also highlighting the impact of in vitro passage on WGS of viral pathogens. Sequencing the whole genome of ILTV was attempted directly from tracheal scrapings of experimentally infected birds to circumvent in vitro culturing. Despite the high number of quality reads obtained from sequencing, assembling the genome was not possible due to poor overlapping sequences and the presence of multiple gaps. A concatenated sequence covering 91% of the ILTV genome was obtained after excluding the regions with low coverage. Further analysis performed on the concatenated genome classified the ILTV isolate as the same class used for infection of the birds (class 9) but revealed the presence of 50 single nucleotide polymorphisms (SNPs) between the two. The results of this study suggest that despite the failure to assemble ILTV genome directly from clinical tissues, the technique has a potential to replace the current PCR-HRM technique used for ILTV typing since it provides far more detailed information about the genome of the ILTV. Thus, the studies reported in this thesis have served as a proof of concept and have contributed to the evaluation of the suitability of WGS as a tool for accurate strain identification directly from clinical specimens of these pathogens. Also, this thesis provided insights into the origin of aHEV isolates currently circulating in Australia.