Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence.

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.

Lumpy skin disease virus (LSDV) causes a disease of economic importance affecting cattle. Its global epidemiology is complex due to the combination of vector-borne and anthropogenic spread, the circulation of vaccine-like recombinants, and the use of vaccines. The slow molecular evolution of its DNA genome limits the utility of genetic variation for accurate tracing based on evolutionary analyses, but this limitation has not yet been formally assessed. Furthermore, until present, whole genome sequencing in affected areas has remained patchy. This study combines the first fine-grained sampling of LSDV whole genomes from a time-constrained (2015–2017) southeastern European (SEE) LSDV outbreak, which we analyze along with curated public genomes to investigate the global and regional viral dispersal dynamics. First, haplotype networks visualizing the limited genetic variability associated with the SEE LSDV outbreak show intense intermixing between countries. We also assess at which spatial scale a correlation between genetic and geographic distances can be detected for LSDV. On a global scale, we show the importance of accounting for recombination events that can impact phylogenetic and phylogeographic reconstructions. Following the assessment of the temporal signal in the recombination-free alignment, our time-scaled continuous phylogeographic analysis of Kenya-like and recent wild-type viruses confirms the origin and global dissemination history of LSDV. Our analyses highlight the importance of careful selection and application of phylodynamic approaches to DNA viruses, as well as the importance of whole genome sampling in endemic and outbreak areas to improve our understanding of the evolution, epidemiology, and transmission dynamics of DNA viruses. IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015–2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Metagenomic next generation sequencing (mNGS) is increasingly recognized as an important complementary tool to targeted human and animal infectious disease diagnostics. It is, however, sensitive to biases and errors that are currently not systematically evaluated by the implementation of quality controls (QC) for the diagnostic use of mNGS. We evaluated a commercial reagent (Mengovirus extraction control kit, CeraamTools, bioMérieux) as an exogenous internal control for mNGS. It validates the integrity of reagents and workflow, the efficient isolation of viral nucleic acids and the absence of inhibitors in individual samples (verified using a specific qRT-PCR). Moreover, it validates the efficient generation of viral sequence data in individual samples (verified by normalized mengoviral read counts in the metagenomic analysis). We show that when using a completely random metagenomics workflow: (1) Mengovirus RNA can be reproducibly detected in different animal sample types (swine feces and sera, wild bird cloacal swabs), except for tissue samples (swine lung); (2) the Mengovirus control kit does not contain other contaminating viruses that may affect metagenomic experiments (using a cutoff of minimum 1 Kraken classified read per million (RPM)); (3) the addition of 2.17 × 106Mengovirus copies/mL of sample does not affect the virome composition of pig fecal samples or wild bird cloacal swab samples; (4) Mengovirus Cq values (using as cutoff the upper limit of the 99 % confidence interval of Cq values for a given sample matrix) allow the identification of samples with poor viral RNA extraction or high inhibitor load; (5) Mengovirus normalized read counts (cutoff RPM > 1) allow the identification of samples where the viral sequences are outcompeted by host or bacterial target sequences in the random metagenomic workflow. The implementation of two QC testing points, a first one after RNA extraction (Mengoviral qRT-PCR) and a second one after metagenomic data analysis provide valuable information for the validation of individual samples and results. Their implementation in addition to external controls validating runs or experiments should be carefully considered for a given sample type and workflow.

HPAI A(H5N1) Clade 2.3.4.4b Genotype EA-2023-DG

The Kachia Grazing Reserve (KGR) is located in Kaduna state in north‐western Nigeria and consists of 6 contiguous blocks housing 744 defined households (HH), all engaged in livestock keeping. It is considered as a homogenous epidemiological unit and a defined study area. In 2012, all cattle and sheep of 40 selected HH were sampled to determine sero‐prevalence of antibodies to foot‐and‐mouth disease virus (FMDV) and of FMDV. The overall sero‐prevalence of antibodies to the non‐structural 3ABC protein (NSP‐3ABC ELISA) was 28.9% (380/1315) (30.6% cattle; 16.3% sheep), and in 4.5% (62/1380) (5% cattle; 0.6% sheep) of the examined sera FMD viral RNA could be detected by real‐time RT‐PCR (rRT‐PCR). Additionally, in 2012 and 2014 serum, epithelium and probang samples were collected from cattle in reported FMD outbreaks and the causative FMDVs were molecularly characterized. Approximately half (28/59) of the outbreak sera reacted positive in NSP‐3ABC ELISA, and 88% (52/59) of the outbreak sera contained detectable viral RNA. Overall, antibodies against five FMDV serotypes (O, A, SAT1, SAT2 and SAT3) were detected by solid phase competitive ELISA with combinations of two or more serotypes being common. Of the 21 FMDVs that could be isolated 19 were sequenced and 18 were confirmed as SAT2 (lineage VII) while one was characterized as serotype O (EA‐3 topotype). Phylogenetic analysis revealed close relationship between Nigerian FMDV strains and strains in this region and even with strains in North‐Africa. Our findings indicate that FMD constitutes an endemic health problem to cattle rearing in the agro‐pastoralist community in the KGR and that the KGR is not a closed epidemiological unit. Insight into the local FMDV epidemiology and in the circulating FMDV serotypes/strains is of support to the relevant authorities in Nigeria when considering the need for an FMD control policy to improve animal production in grazing reserves.