Edoardo Giussani

This study uses a phylodynamic approach to investigate host species contribution in the spatiotemporal dynamics of the highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b identified during the 2024–2025 epidemic in Italy. Our findings revealed the key role of swans in the initial virus incursions, the central role of synanthropic bird species as the bridge hosts at the wild‐domestic interface, and provided evidence of sustained circulation in both wild and domestic birds of viruses carrying mammalian‐adaptive mutations. Overall, our results highlight a complex, multi‐host transmission dynamic at the wildlife–domestic interface and underscore the value of a One Health, genomics‐informed surveillance approach, explicitly including synanthropic species, to reconstruct transmission pathways and refine control strategies.

The 2021–2022 high pathogenicity avian influenza (HPAI) epizootic was the worst ever recorded in Europe in general and in Spain in particular. Between December 2021 and November 2022, H5N1 clade 2.3.4.4b HPAI viruses caused outbreaks in both wild birds and domestic poultry in Spain. We analysed the complete genome sequences of H5N1 HPAI viruses identified during this period in Spain and conducted comparative phylogenetic analyses to identify their origin and reconstruct their evolutionary and diffusion dynamics. We identified four different genetic reassortants of H5N1 clade 2.3.4.4b HPAI viruses. Our results suggest multiple wild bird introductions of H5N1 clade 2.3.4.4b HPAI viruses into different regions of Spain from other European countries. Bayesian phylodynamic analyses of H5N1 clade 2.3.4.4b HPAI viruses support that their initial entry into Spain occurred in the North-West and South-West through wild birds, which further spread the viruses to other regions within Spain. Andalusia (South) was the hotspot for maintenance of viruses in poultry. Wild Anseriformes played a crucial role in the introduction of the viruses into Spain and the subsequent transmission of these viruses to other host types of birds, both wild and domestic. This study highlights the role of wild birds in the ecology of H5N1 clade 2.3.4.4b HPAI viruses and provides further insight into the genetic diversity, evolution, and spread of these viruses between wild birds and poultry.

Over the past century, Influenza A virus (IAV) has caused four of the five reported pandemics, all of which originated from viruses possessing genome segments of avian origin. The recent spread of highly pathogenic avian influenza (HPAI) viruses, particularly the clade 2.3.4.4b A(H5N1) subtype, has led to an alarming increase in mammalian infections, raising concerns about the potential for future pandemics. In response to this, we developed FluMut, an open-source, cross-platform tool designed to identify molecular markers with potential impacts on H5N1 virus phenotypes. FluMut leverages an up-to-date database, FluMutDB, to rapidly analyze thousands of nucleotide sequences, identifying mutations associated with host adaptation, increased virulence, and antiviral resistance. The tool is available both as a command-line interface and a user-friendly graphical interface, making it accessible to researchers with varying levels of computational expertise. FluMut provides comprehensive outputs, including tables of detected markers, their biological effects, and corresponding literature references. This tool fills a critical gap in the genomic surveillance of HPAI H5N1, facilitating real-time monitoring of viral evolution and aiding in the identification of mutations that may signal increased pandemic potential. Future updates will extend FluMut’s capabilities to other influenza subtypes.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020–2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

The SARS-CoV-2 Delta variant of concern (VOC) was often associated with serious clinical course of the COVID-19 disease. Herein, we investigated the selective pressure, gene flow and evaluation on the frequencies of mutations causing amino acid substitutions in the Delta variant in three Italian regions. A total of 1500 SARS-CoV-2 Delta genomes, collected in Italy from April to October 2021 were investigated, including a subset of 596 from three Italian regions. The selective pressure and the frequency of amino acid substitutions and the prediction of their possible impact on the stability of the proteins were investigated. Delta variant dataset, in this study, identified 68 sites under positive selection: 16 in the spike (23.5%), 11 in nsp2 (16.2%) and 10 in nsp12 (14.7%) genes. Three of the positive sites in the spike were located in the receptor-binding domain (RBD). In Delta genomes from the three regions, 6 changes were identified as very common (>83.7%), 4 as common (>64.0%), 21 at low frequency (2.1%–25.0%) and 29 rare (≤2.0%). The detection of positive selection on key mutations may represent a model to identify recurrent signature mutations of the virus.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on 19 May 2021. The outbreak consisted of three phases: May–June 2021, September–November 2021, and January–May 2022. In total, 32 backyards and 10 commercial holdings tested positive for the virus. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that had been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of three H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on May 19, 2021. The outbreak consisted of three temporal phases: May–June 2021, September–November 2021, and January–May 2022. In total, 32 backyard and 10 commercial holdings tested positive for the virus, affecting 179,198 poultry. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that have been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of 3 H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.

In 2021, amidst the COVID-19 pandemic and global food insecurity, the Nigerian poultry sector was exposed to the highly pathogenic avian influenza (HPAI) virus and its economic challenges. Between 2021 and 2022, HPAI caused 467 outbreaks reported in 31 of the 37 administrative regions in Nigeria. In this study, we characterized the genomes of 97 influenza A viruses of the subtypes H5N1, H5N2, and H5N8, which were identified in different agro-ecological zones and farms during the 2021–2022 epidemic. The phylogenetic analysis of the HA genes showed a widespread distribution of the H5Nx clade 2.3.4.4b and similarity with the HPAI H5Nx viruses that have been detected in Europe since late 2020. The topology of the phylogenetic trees indicated the occurrence of several independent introductions of the virus into the country, followed by a regional evolution of the virus that was most probably linked to its persistent circulation in West African territories. Additional evidence of the evolutionary potential of the HPAI viruses circulating in this region is the identification in this study of a putative H5N1/H9N2 reassortant virus in a mixed-species commercial poultry farm. Our data confirm Nigeria as a crucial hotspot for HPAI virus introduction from the Eurasian territories and reveal a dynamic pattern of avian influenza virus evolution within the Nigerian poultry population.

In 2021, amidst the COVID-19 pandemic and global food insecurity, the Nigerian poultry sector was yet exposed to highly pathogenic avian influenza (HPAI) virus and its economic challenges. Between 2021 and 2022, HPAI caused 467 outbreaks reported in 31 of the 37 administrative regions in Nigeria. In this study, we characterized the genome of 97 influenza A viruses of the subtypes H5N1, H5N2 and H5N8 identified in different agro-ecological zones and farms during the 2021-2022 epidemic. The phylogenetic analysis of the HA genes showed widespread distribution of the H5Nx clade 2.3.4.4b and similarity with the HPAI H5Nx viruses detected in Europe since late 2020. Topology of the phylogenetic trees indicates the occurrence of several independent introductions of the virus into the country followed by a regional evolution of the virus most probably linked to its persistent circulation in West African territories. An additional evidence of the evolutionary potential of HPAI viruses circulating in this region is the identification in this study of a putative H5N1/H9N2 reassortant virus in a mixed-species commercial poultry farm. Our data confirm Nigeria as a crucial hotspot for HPAI virus introduction from the Eurasian territories and reveal a dynamic pattern of avian influenza virus evolution within the Nigerian poultry population.

In October 2022, an outbreak in Europe of highly pathogenic avian influenza (HPAI) A(H5N1) in intensively farmed minks occurred in northwest Spain. A single mink farm hosting more than 50,000 minks was involved. The identified viruses belong to clade 2.3.4.4b, which is responsible of the ongoing epizootic in Europe. An uncommon mutation (T271A) in the PB2 gene with potential public health implications was found. Our investigations indicate onward mink transmission of the virus may have occurred in the affected farm.