High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Tracking the Selective Pressure Profile and Gene Flow of SARS-CoV-2 Delta Variant in Italy from April to October 2021 and Frequencies of Key Mutations from Three Representative Italian Regions.

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.

Emergence and Persistent Circulation of Highly Pathogenic Avian Influenza Virus A (H5N8) in Kosovo, May 2021-May 2022.

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.

The Evolution of Highly Pathogenic Avian Influenza A (H5) in Poultry in Nigeria, 2021-2022.

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.

Highly pathogenic avian influenza A (H5N1) virus infections in wild carnivores connected to mass mortalities of pheasants in Finland.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe during 2020-2022. Virus types H5N8 and H5N1 have dominated the epidemic. Isolated spill-over infections in mammals started to emerge as the epidemic continued. In autumn 2021, HPAI H5N1 caused a series of mass mortality events in farmed and released pheasants (Phasianus colchicus) in a restricted area in southern Finland. Later, in the same area, an otter (Lutra lutra), two red foxes (Vulpes vulpes) and a lynx (Lynx lynx) were found moribund or dead and infected with H5N1 HPAI virus. Phylogenetically, H5N1 strains from pheasants and mammals clustered together. Molecular analyses of the four mammalian virus strains revealed mutations in the PB2 gene segment (PB2-E627K and PB2-D701N) that are known to facilitate viral replication in mammals. This study revealed that avian influenza cases in mammals were spatially and temporally connected with avian mass mortalities suggesting increased infection pressure from birds to mammals.

Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022.

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.

Emergence of a Reassortant 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Containing H9N2 PA Gene in Burkina Faso, West Africa, in 2021.

Since 2006, the poultry population in Burkina Faso has been seriously hit by different waves of Highly Pathogenic Avian Influenza (HPAI) H5N1 epizootics. In December 2021, three distinct regions of Burkina Faso, namely, Gomboussougou, Bonyollo, and Koubri, detected HPAI H5N1 viruses in poultry. Whole genome characterization and statistical phylogenetic approaches were applied to shed light on the potential origin of these viruses and estimate the time of virus emergence. Our results revealed that the HPAI H5N1 viruses reported in the three affected regions of Burkina Faso cluster together within clade 2.3.4.4b, and are closely related to HPAI H5N1 viruses identified in Nigeria and Niger in the period 2021-2022, except for the PA gene, which clusters with H9N2 viruses of the zoonotic G1 lineage collected in West Africa between 2017 and 2020. These reassortant viruses possess several mutations that may be associated with an increased zoonotic potential. Although it is difficult to ascertain where and when the reassortment event occurred, the emergence of a H5N1/H9N2 reassortant virus in a vulnerable region, such as West Africa, raises concerns about its possible impact on animal and human health. These findings also highlight the risk that West Africa may become a new hotspot for the emergence of new genotypes of HPAI viruses.

SARS-CoV-2 intra-host evolution during prolonged infection in an immunocompromised patient.

OBJECTIVES: Intra-host SARS-CoV-2 evolution during chronic infection in immunocompromised hosts has been suggested as being the possible trigger of the emergence of new variants. METHODS: Using a deep sequencing approach, we investigated the SARS-CoV-2 intra-host genetic evolution in a patient with HIV over a period of 109 days. RESULTS: Sequencing of nasopharyngeal swabs at three time points demonstrated dynamic changes in the viral population, with the emergence of 26 amino acid mutations and two deletions, 57% of them in the Spike protein. Such a combination of mutations has never been observed in other SARS-CoV-2 lineages detected so far. CONCLUSION: Our data confirm that persistent infection in certain immunocompromised individuals for a long time may favor the dangerous emergence of new SARS-CoV-2 variants with immune evasion properties.

Spatiotemporal Dynamics, Evolutionary History and Zoonotic Potential of Moroccan H9N2 Avian Influenza Viruses from 2016 to 2021.

The H9N2 virus continues to spread in wild birds and poultry worldwide. At the beginning of 2016, the H9N2 Avian influenza virus (AIV) was detected in Morocco for the first time; despite the implementation of vaccination strategies to control the disease, the virus has become endemic in poultry in the country. The present study was carried out to investigate the origins, zoonotic potential, as well as the impact of vaccination on the molecular evolution of Moroccan H9N2 viruses. Twenty-eight (28) H9N2 viruses collected from 2016 to 2021 in Moroccan poultry flocks were isolated and their whole genomes sequenced. Phylogenetic and evolutionary analyses showed that Moroccan H9N2 viruses belong to the G1-like lineage and are closely related to viruses isolated in Africa and the Middle East. A high similarity among all the 2016-2017 hemagglutinin sequences was observed, while the viruses identified in 2018-2019 and 2020-2021 were separated from their 2016-2017 ancestors by long branches. Mutations in the HA protein associated with antigenic drift and increased zoonotic potential were also found. The Bayesian phylogeographic analyses revealed the Middle East as being the region where the Moroccan H9N2 virus may have originated, before spreading to the other African countries. Our study is the first comprehensive analysis of the evolutionary history of the H9N2 viruses in the country, highlighting their zoonotic potential and pointing out the importance of implementing effective monitoring systems.

Molecular Monitoring of SARS-CoV-2 in Different Sewage Plants in Venice and the Implications for Genetic Surveillance.

Wastewater-based epidemiology is now widely used as an indirect tool to monitor the spread of SARS-CoV-2. In this study, five different sample matrices representing diverse phases of the wastewater treatment process were collected during the second wave of SARS-CoV-2 from two wastewater treatment plants (WWTPs) serving the Civil Hospital and Sacca Fisola island in Venice, Italy. Positive SARS-CoV-2 detections occurred at both WWTPs, and data on viral genome detection rate and quantification suggest that the pellet (i.e., the particulate resulting from the influent) is a sensitive matrix that permits reliable assessment of infection prevalence while reducing time to results. On the contrary, analysis of post-treatment matrices provides evidence of the decontamination efficacy of both WWTPs. Finally, direct sequencing of wastewater samples enabled us to identify B.1.177 and B.1.160 as the prevalent SARS-CoV-2 lineages circulating in Venice at the time of sampling. This study confirmed the suitability of wastewater testing for studying SARS-CoV-2 circulation and established a simplified workflow for the prompt detection and characterization of the virus.

A high definition picture of somatic mutations in chronic lymphoproliferative disorder of natural killer cells.

The molecular pathogenesis of chronic lymphoproliferative disorder of natural killer (NK) cells (CLPD-NK) is poorly understood. Following the screening of 57 CLPD-NK patients, only five presented STAT3 mutations. WES profiling of 13 cases negative for STAT3/STAT5B mutations uncovered an average of 18 clonal, population rare and deleterious somatic variants per patient. The mutational landscape of CLPD-NK showed that most patients carry a heavy mutational burden, with major and subclonal deleterious mutations co-existing in the leukemic clone. Somatic mutations hit genes wired to cancer proliferation, survival, and migration pathways, in the first place Ras/MAPK, PI3K-AKT, in addition to JAK/STAT (PIK3R1 and PTK2). We confirmed variants with putative driver role of MAP10, MPZL1, RPS6KA1, SETD1B, TAOK2, TMEM127, and TNFRSF1A genes, and of genes linked to viral infections (DDX3X and RSF1) and DNA repair (PAXIP1). A truncating mutation of the epigenetic regulator TET2 and a variant likely abrogating PIK3R1-negative regulatory activity were validated. This study significantly furthered the view of the genes and pathways involved in CLPD-NK, indicated similarities with aggressive diseases of NK cells and detected mutated genes targetable by approved drugs, being a step forward to personalized precision medicine for CLPD-NK patients.

Lack of Viral Load Within Chronic Lymphoproliferative Disorder of Natural Killer Cells: What Is Outside the Leukemic Clone?

Large granular lymphocyte leukemias (LGLL) are sustained by proliferating cytotoxic T cells or NK cells, as happens in Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK), whose etiology is only partly understood. Different hypotheses have been proposed on the original events triggering NK cell hyperactivation and transformation, including a role of viral agents. In this perspective, we revise the lines of evidence that suggested a pathogenetic role in LGLL of the exposure to retroviruses and that identified Epstein Barr Virus (EBV) in other NK cell leukemias and lymphomas and focus on the contrasting data about the importance of viral agents in CLPD-NK. EBV was detected in aggressive NK leukemias but not in the indolent CLPD-NK, where seroreactivity against HTLV-1 retrovirus envelope BA21 protein antigens has been reported in patients, although lacking clear evidence of HTLV infection. We next present original results of whole exome sequencing data analysis that failed to identify viral sequences in CLPD-NK. We recently demonstrated that proliferating NK cells of patients harbor several somatic lesions likely contributing to sustain NK cell proliferation. Thus, we explore whether "neoantigens" similar to the BA21 antigen could be generated by aberrancies present in the leukemic clone. In light of the literature and new data, we evaluated the intriguing hypothesis that NK cell activation can be caused by retroviral agents located outside the hematopoietic compartment and on the possible mechanisms involved with the prospects of immunotherapy-based approaches to limit the growth of NK cells in CLPD-NK disease.