Biology subjects

Tennakoon, C.

Publications and source records attributed to Tennakoon, C..

6 recordsLinked to original sources

bioRxiv2026

First detection of High pathogenicity Avian Influenza A(H5N1) Clade 2.3.4.4b Genotype EA-2024-DI.2.1 in Egypt associated with migratory wild birds

High pathogenicity avian influenza (HPAI) H5 clade 2.3.4.4b is the main driver of the ongoing unprecedented global panzootic. The recently emerged HPAI H5N1 clade 2.3.4.4b genotype EA-2024-DI.2.1 has become predominant in Europe, with migratory wild birds, particularly waterfowl, playing a major role in its dissemination. Egypt lies along major Afro-Eurasian migratory flyways, which have historically played an important role in the introduction of emerging H5Nx viruses into the country. In this study, targeted surveillance was conducted on 416 wild birds offered for sale in in live bird markets (LBMs) and roadside trading points in northern Egypt, mainly in Damietta and Port Said. Of these, 118 birds showing mild clinical signs were examined post-mortem and lung and tracheal tissues were collected, while oropharyngeal and cloacal swabs were collected from apparently healthy birds. Avian influenza virus was detected by RT-qPCR in 22 wild birds, all from tissue samples, whereas all swabs from apparently healthy birds were negative. Waterfowl accounted for 16 of the 22 positive birds (72.7%), with Eurasian teal showing the lowest Ct values (21-25). Phylogenetic and whole-genome analyses showed that the sequenced wild-bird viruses clustered within the recently emerged EA-2024-DI.2.1 sub-lineage and were closely related to contemporary European viruses. Compared with the EA-2021-AB genotype currently circulating in Egyptian poultry, the EA-2024-DI.2.1 viruses showed several HA amino acid differences, including A83D, L104M and T195A. These findings provide evidence for the introduction of EA-2024-DI.2.1 into Egypt through migratory wild birds and highlight the importance of continued genomic surveillance at the wild bird domestic poultry interface and antigenic evaluation against vaccines currently used in Egypt.

molecular biology↗

bioRxiv2025

Using pangenome variation graphs to improve mutation detection in a large DNA virus

Accurately quantifying viral genetic diversity is essential for understanding pathogen evolution, transmission, and emergence. However, standard approaches that map sequencing reads to a single linear reference genome introduce substantial reference bias, particularly for samples that are divergent, recombinant, or belong to rare lineages. Pangenome variation graphs (PVGs) mitigate this issue by representing multiple genomes within a unified graph structure, enabling read mapping across all observed and potential haplotypes. Despite this, PVGs have rarely been applied to viruses. Here, we address this gap by constructing and evaluating the first PVG for lumpy skin disease virus (LSDV), an emerging poxvirus of global importance. We generated PVGs of different sizes and mapped Illumina datasets using Giraffe, benchmarking performance against linear reference mapping with Minimap2. A minimal three-sample PVG containing one representative from each major lineage recovered 97% of known LSDV nucleotide diversity while reducing PVG size by >95% relative to a 121-sample PVG. PVG-based mapping detected more SNPs than linear mapping, including variants supported by read evidence that was not detected when reads were mapped to a single reference genome. 27% of SNPs detected using PVG-based mapping could not be projected onto the linear reference coordinate system because they were on alternative paths absent from reference, highlighting the impacts of reference bias. Notably, these new SNPs were at genes involved in host recognition and immune evasion, and identified lineage-specific mutations that improved subclade phylogenetic structure. Our findings demonstrate that PVGs substantially enhance SNP discovery in LSDV, with direct implications for genomic surveillance, outbreak tracing, and the detection of recombinant vaccine-related lineages in LSDV and other large DNA viruses. Impact StatementGenomic surveillance of viral pathogens typically relies on mapping reads to a single reference genome, a practice that systematically misses variation in divergent or recombinant isolates. We show that pangenome variation graphs (PVGs) can reduce this limitation in DNA viruses. Using lumpy skin disease virus (LSDV) as a model system, we demonstrate that a compact PVG constructed from only three representative genomes captures most known genomic diversity and improves the detection of mutations that are difficult to identify using linear reference mapping. PVGs recover biologically meaningful variants at genes involved in immune evasion and host interaction, resolve closely related subclades, and reveal mutations that cannot be identified using a single reference genome. This work uses a population-structure-guided strategy for building reference viral PVGs that balance computational efficiency with sensitivity, offering clear benefits for genomic surveillance, outbreak reconstruction, and the detection of recombinant lineages. Data SummaryAll supporting code and protocols have been provided within the article or through supplementary data files. Supplementary tables are available at Figshare: https://figshare.com/s/7a879df5b5452d04585b with all code at https://github.com/downingtim/LSDV_pangenomics

genomics↗

bioRxiv2025

Panalyze: automated virus pangenome variation graph construction and analysis

MotivationConstructing and studying pangenome variation graphs (PVG) supports new insights into viral genomic diversity. This is because such pangenomes are less prone to reference bias, which affects mutation detection. Interpreting the information arising from this is challenging, so automating these processes to allow exploratory investigations for PVG optimisation is essential. Moreover, existing methods do not scale well to the smaller virus genome sizes and to facilitate analysis in laptop environments. To address this, we developed an easily deployable pipeline to facilitate the rapid creation of virus PVGs that applies a broad range of analyses to these PVGs. ResultsWe present panalyze, a scalable and unbiased virus PVG construction, analysis and annotation tool implemented in NextFlow and containerised in Docker. Panalyze uses NextFlow to efficiently complete tasks across multiple compute nodes and in diverse high-performance computing environments. Panalyze can also operate on a single thread on a standard laptop, and analyse sequences lengths are of any size. We illustrate how Panalyze works and the valuable outputs it can generate using a range of common viral pathogens. AvailabilityPanalyze is released under a MIT open-source license, available on GitHub with documentation accessible at https://github.com/downingtim/Panalyze/. ContactChandana.tennakoon@pirbright.ac.uk, tim.downing@pibright.ac.uk Supplementary informationSupplementary data are available online.

bioinformatics↗

bioRxiv2025

Prior infection with IBDV prolonged the shedding of a mallard H3N8 influenza A virus (IAV) challenge from the oropharyngeal cavity of some chickens and increased the number of amino acid substitutions in the IAV samples

Infectious bursal disease virus (IBDV) is endemic worldwide and causes immunosuppression in chickens. We hypothesized that a previous history of IBDV in chickens would render them more susceptible to infection by influenza A viruses (IAVs) from aquatic waterfowl reservoirs. To model this, we inoculated 14 day old specific pathogen free (SPF) chickens with a low pathogenicity avian influenza (LPAI) virus strain from a mallard (A/Mallard/Alberta/156/01 (H3N8)) and compared replication and shedding between immunocompetent chickens and chickens that had immune dysregulation due to a prior IBDV infection with strain F52/70 (genogroup A1B1) at 2 days of age. The mallard IAV strain replicated in the upper respiratory tract of the chickens, and virus was shed from the oropharyngeal cavity, but there was no shedding from the cloaca, and no transmission to sentinel chickens. Replication of the mallard IAV in the chicken host was associated with amino acid substitutions in the polymerase complex and HA. IBDV infection increased the average fold change of IAV replication in the trachea of chickens, prolonged the shedding of infectious IAV from 5 to 6 days in some chickens, increased the number of amino acid substitutions detected in the IAV population from 13 to 30, and significantly increased the number of mutations per IAV sample from 2.50 (SD +/- 1.83) in the Mock/IAV group to 4.75 (SD +/- 1.81) in the IBDV/IAV group (p < 0.01). Taken together, IBDV infection prolonged the shedding of the mallard IAV in some chickens and changed IAV intra-host evolution. Author summarySpillover of IAVs from wild aquatic waterfowl into poultry populations occur frequently, which increases the risk of human infection as people have more contact with poultry than wild birds. Poultry flocks may have other co-morbidities that may influence the spread of IAV. Our data demonstrate that prior IBDV infection increased the average fold change of a mallard H3N8 LPAI virus in the trachea of inoculated chickens, prolonged the shedding of infectious IAV from the oropharyngeal cavity, and significantly increased the average number of amino acid substitutions per IAV sample. We hypothesize that IBDV infection could increase the amount of IAV shed into the environment and broaden the diversity of the IAV population shed. We conclude that controlling the spread of wild aquatic waterfowl strains of IAV in chickens should involve a holistic approach, including the control of co-morbidities and immunosuppressive diseases that could exacerbate their spread.

microbiology↗

bioRxiv2023

Full genome sequence analysis of African swine fever virus isolates from Cameroon

African swine fever is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of ASFV is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.

microbiology↗

bioRxiv2021

A random priming amplification method for whole genome sequencing of SARS-CoV-2 and H1N1 influenza A virus.

BackgroundNon-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. MethodsThe performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. ResultsThe SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of COVID that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. ConclusionsThe SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

microbiology↗