African Swine Fever (ASF) is an important contagious haemorrhagic viral disease affecting swine whose notification is mandatory due to its high mortality rates and the great sanitary and socioeconomic impact it has on international trade in animal and swine products. This disease only affects porcine species, both wild and domestic, and produces a variety of clinical signs such as fever and functional disorders of the digestive and respiratory systems. Lesions are mainly characterized by congestive-haemorrhagic alterations. ASF epidemiology varies significantly between countries, regions and continents, since it depends on the characteristics of the virus in circulation, the presence of wild hosts and reservoirs, environmental conditions and human social behaviour. Furthermore, a specific host will not necessarily always play the same active role in the spread and maintenance of ASF in a particular area. Currently, ASF is endemic in most sub-Saharan African countries where wild hosts and tick vectors (Ornithodoros) play an important role acting as biological reservoirs for the virus. In Europe, the disease has been endemic since 1978 on the island of Sardinia (Italy) and since 2007, when it was first reported in Georgia, in a number of Eastern European countries. It is also endemic in certain regions of the Russia Federation, where domestic pig and wild boar populations are widely affected. By contrast, in the affected eastern European Union (EU) countries where ASF is currently as epidemic, the on-going spread of the disease affects mainly wild boar populations located in restricted areas and, to a much less extent, domestic pigs. Unlike most livestock diseases, no vaccine or specific treatment is currently available for ASF. Therefore, disease control is mainly based on early detection and the application of strict sanitary and biosecurity measures. Epidemiology of ASF is very complex by the existence of different virus circulating, reservoirs and a number of scenarios, and the on-going spread of the disease through Africa and Europe. Survivor pigs can remain persistently infected for months which may contribute to virus transmission and thus the spread and maintenance of the disease, thereby complicating attempts to control it.

A highly sensitive and specific real‐time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n = 260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real‐time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV‐infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous β‐actin gene. Finally, the complete study offers a new validated real‐time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF.

Bagaza virus (BAGV) is a mosquito-borne flavivirus belonging to the Ntaya serocomplex. In 2010, a disease outbreak was reported in Cádiz (Southern Spain) affecting game birds (red-legged partridges and common pheasants). In this work, red-legged partridges were inoculated experimentally with infectious BAGV isolated from this outbreak in order to make a complete clinical and analytical assessment of the disease caused by the pathogen in this species. Viral load (by real-time RT-PCR) in blood, oral and cloacal swabs, and feathers, and neutralizing antibody titres (by VNT) were measured. In order to determine direct contact transmission, non-inoculated partridges were caged together with the inoculated ones. To assess infectiousness in other species, house sparrows and mice were also inoculated with the virus. All the inoculated partridges were clinically affected, and 30% of them died. All the infected individuals lost weight, with larger losses being recorded in females. Conversely, no mortality or disease symptoms were observed in the sparrows or mice. Remarkably, all the contact partridges acquired the infection by direct (non-vectored) transmission. This study confirms that the red-legged partridge is a susceptible host for BAGV infection, and that this pathogen is transmitted by direct contact. Long-lasting viral loads detected in calami of immature feathers demonstrate that feather sampling could be a useful strategy in active surveillance programs for early detection of BAGV.

Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared the results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and a Rapid Barcoding Kit (RBK) vs a Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in thymine and adenine homopolymeric regions than the R9, at the expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than the RBK, and provided longer reads than the RBK. It also resulted in a larger percentage of aligned reads (99 vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK preparation library provided longer DNA fragments which contributed to a better assembly of the SARS-CoV-2, despite an impaired detection of variants in a R10 flow cell. Nanopore sequencing could be used in epidemiological surveillance of SARS-CoV-2. Key points • Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance. • Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome. • Ligation sequencing is preferred rather than the rapid kit using transposases.

West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach.