Chandana Tennakoon

Infectious bronchitis virus (IBV), a Gammacoronavirus , causes the economically damaging disease of poultry, infectious bronchitis. Development of IBV live attenuated vaccines (LAVs) is dependent on the supply of embryonated hens’ eggs and attenuation through serial passaging, upwards of 80 times. Vaccine development needs to keep pace with IBV evolution; an issue compounded as cross protection between individual IBV serotypes is unpredictable and often limited. Rational attenuation alongside the ability to propagate LAVs in cell culture offers significant advantages in terms of adaptability, cost, and speed. Most field IBV strains exhibit restricted in vitro tropism, being unable to replicate in primary or continuous cells without serial passage and adaptation. Beaudette, an attenuated IBV strain, is capable of replication in Vero cells, an ability conferred by the spike (S) glycoprotein. In this study, utilizing a recombinant IBV based on the nephropathogenic IBV strain D388, in which the S sequence is replaced with the equivalent sequence derived from Beaudette, we determine that the Beaudette spike can confer the ability to replicate in Vero cells and Beaudette’s associated attenuated in vivo phenotype to a distantly related IBV strain. Assessment of infectious progeny and viral RNA in vivo suggests that attenuation was conferred by a tissue tropism-independent molecular mechanism. Analysis of innate immune responses revealed a lower intensity and less inflammatory profile. Our findings support the use of the Beaudette S protein as an avenue for the development of rationally attenuated, cell culture-propagated rIBV vaccines and identify the S protein as a contributor to nephropathogenicity. IMPORTANCE: Infectious bronchitis virus (IBV), a Gammacoronavirus of chickens, causes significant economic losses worldwide. Current live attenuated vaccines (LAVs) are generated through extensive serial passage of virulent IBV strains in specific-pathogen-free (SPF) embryonated hen’s eggs, upwards of 80 passages. This process is slow, dependent on the availability of SPF embryonated eggs, and the molecular basis of attenuation remains poorly understood. Development of rationally attenuated, cell-culture-based IBV vaccines would enable more rapid responses to emerging viral variants/strains. Using a recombinant IBV, we demonstrated the spike glycoprotein from the attenuated IBV strain Beaudette can confer attenuation and the ability to replicate in Vero cells to a distantly related virulent IBV strain. Attenuation was not linked to changes in tissue tropism but was associated with altered host responses. Our research provides new insights into IBV pathogenesis and presents an avenue for rational attenuation that can be exploited for future development of cell-based IBV vaccines.

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 and 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. Twenty-seven per cent 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 the 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.

Infectious bursal disease virus (IBDV) causes an endemic immunosuppressive disease in chickens. Prior exposure to IBDV influences the pathogenesis and shedding of chicken strains of influenza A virus (IAV), but its effect on waterfowl strains is poorly understood. To address this, we inoculated 14-day-old specific pathogen-free chickens with low pathogenicity avian influenza strain A/Mallard/Alberta/156/01 (H3N8) and compared the replication, shedding, pathogenesis, transmission and intra-host evolution between immunocompetent chickens and chickens that had IBDV-mediated immune dysregulation due to a prior infection with strain F52/70 at 2 days of age. The IAV replicated in the upper respiratory tract, and the virus was shed from the oropharyngeal cavity, but there was no shedding from the cloaca and no transmission to sentinel chickens. IAV replication in chickens was associated with amino acid substitutions in the polymerase complex and HA. Prior IBDV infection had no significant effect on IAV pathogenicity, replication or shedding and had a modest effect on IAV diversity, increasing the number of amino acid substitutions from an average of 2.50 substitutions per sample ( sd ±1.83) in the Mock/IAV group to 4.75 ( sd ±1.81) in the IBDV/IAV group ( P <0.01). Taken together, our data suggest that IBDV is unlikely to play a major role in the spillover or spread of waterfowl IAV strains in chicken flocks, although it could expand IAV diversity. This information is useful for informing preventative measures for controlling IAV in poultry flocks.

Motivation: Constructing and studying pangenome variation graphs (PVGs) 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. Results: We present Panalyze, a computationally scalable 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 computing environments. Panalyze can also operate on a single thread on a standard laptop, and analyse sequence lengths of any size. We illustrate how Panalyze works and the valuable outputs it can generate using a range of common viral pathogens. Availability and implementation: Panalyze is released under a MIT open-source license, available on GitHub with documentation accessible at https://github.com/downingtim/Panalyze/.

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 Statement: Genomic 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 Summary: All 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

Motivation Constructing and studying reference-free pangenome variation graphs (PVG) can support new insights into viral genomic diversity. Interpreting the information arising from this is challenging, so automating these processes to allow exploratory investigations for optimisation is essential. To address this, we developed an easily deployable pipeline to facilitate the rapid creation of virus pangenome variation graphs that applies a broad range of analyses to these graphs. Results We present panalyze, a scalable and unbiased virus pangenome variation graph construction and analysis tool implemented in NextFlow and containerised in Docker. Panalyze avails of NextFlow to efficiently complete tasks across multiple compute nodes and in diverse High-Performance Computing (HPC) environments. We illustrate how Panalyze works and the valuable outputs it can generate using a range of common viral pathogens Availability Panalyze is released under a MIT open-source license, available on GitHub with documentation accessible at https://github.com/downingtim/LSDV-PVG/. Supplementary information Supplementary data are available at Bioinformatics online.

Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples (“Neethling-like” clade 1.1 and “Kenya-like” subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

Foot-and-mouth disease (FMD) is endemic in large parts of sub-Saharan Africa, Asia and South America, where outbreaks in cloven-hooved livestock threaten food security and have severe economic impacts. Vaccination in endemic regions remains the most effective control strategy. Current FMD vaccines are produced from chemically inactivated foot-and-mouth disease virus (FMDV) grown in suspension cultures of baby hamster kidney 21 cells (BHK-21). Strain diversity means vaccines produced from one subtype may not fully protect against circulating disparate subtypes, necessitating the development of new vaccine strains that “antigenically match”. However, some viruses have proven difficult to adapt to cell culture, slowing the manufacturing process, reducing vaccine yield and limiting the availability of effective vaccines, as well as potentiating the selection of undesired antigenic changes. To circumvent the need to cell culture adapt FMDV, we have used a systematic approach to develop recombinant suspension BHK-21 that stably express the key FMDV receptor integrin αvβ6. We show that αvβ6 expression is retained at consistently high levels as a mixed cell population and as a clonal cell line. Following exposure to field strains of FMDV, these recombinant BHK-21 facilitated higher virus yields compared to both parental and control BHK-21, whilst demonstrating comparable growth kinetics. The presented data supports the application of these recombinant αvβ6-expressing BHK-21 in future FMD vaccine production.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.