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In November 2024, an outbreak of highly pathogenic avian influenza (HPAI) H5N1 was confirmed in backyard poultry in Timiș County, Western Romania. The index cases involved chickens and domestic geese found dead with lesions characteristic of HPAI. Laboratory confirmation was achieved by real-time RT-qPCR targeting the matrix, H5, and N1 genes, followed by virus isolation in embryonated specific-pathogen-free eggs. Sequencing of the hemagglutinin cleavage site revealed the multi-basic motif PLREKRRKR/GLFG, consistent with a highly pathogenic phenotype. To investigate potential viral persistence, tracheal and cloacal swabs were collected from apparently healthy selected backyard poultry (chickens, geese, ducks and pheasants). RNA extraction and RT-qPCR, performed using protocols validated by the European Union Reference Laboratory for Avian Influenza, yielded negative results for all samples. Internal controls confirmed assay reliability, excluding the possibility of PCR inhibition. The investigation confirmed the occurrence of HPAI H5N1 in backyard poultry and demonstrated the absence of detectable viral persistence in surrounding flocks under the tested conditions. These findings highlight the importance of rapid molecular diagnostics, active surveillance, and strict biosecurity in limiting virus spread. Continued monitoring under the One Health framework is essential to mitigate the risk of avian influenza at the human–animal–environment interface. Full article

In the winter of 2023/2024, the mass death of swans was observed on Lake Karakol on the eastern coast of the Caspian Sea. From 21 December 2023 to 25 January 2024, 1132 swan corpses (Cygnus olor, Cygnus cygnus) were collected and disposed of on the coast by veterinary services and ecologists. Biological samples were collected from 18 birds for analysis at different dates of the epizootic. It was found that the influenza outbreak was associated with a high concentration of migrating birds at Lake Karakol as a result of a sharp cold snap in the northern regions. At different dates of the epizootic, three avian influenza A/H5N1 viruses of clade 2.3.4.4.b were isolated from dead birds and identified as highly pathogenic viruses (HPAIs) based on the amino acid sequence of the hemagglutinin multi-base proteolytic cleavage site (PLREKRRRKR/G). A phylogenetic analysis showed that the viruses isolated from the swans had reassortations in the PB2, PB1, and NP genes between highly pathogenic (HP) and low-pathogenic (LP) avian influenza viruses. Avian influenza viruses A/Cygnus cygnus/Karakol lake/01/2024(H5N1) and A/Mute swan/Karakol lake/02/2024(H5N1) isolated on 10 January 2024 received PB2, PB1, and NP from LPAIV, while A/Mute swan/Mangystau/9809/2023(H5N1) isolated on 26 December 2023 received PB1 and NP from LPAIV, indicating that the H5N1 viruses in this study are new reassortants. All viruses showed amino acid substitutions in the PB2, PB1, NP, and NS1 segments, which are critical for enhanced virulence or adaptation in mammals. An analysis of the genomes of the isolated viruses showed that bird deaths during different periods of the epizootic were caused by different reassortant viruses. Kazakhstan is located at the crossroads of several migratory routes of migratory birds, and the possible participation of wild birds in the introduction of various pathogens into the regions of Kazakhstan requires further study. Full article

Comprehending the replication kinetics of SARS-CoV-2 variants helps explain why certain variants spread more easily, are more contagious, and pose a significant health menace to global populations. The replication kinetics of the Malaysian isolates of Alpha, Beta, Delta, and Omicron variants were studied in the Vero E6 cell line. Their replication kinetics were determined using the plaque assay, quantitative real-time PCR (qRT-PCR), and the viral growth curve. The Beta variant exhibited the highest replication rate at 24 h post-infection (h.p.i), as evidenced by the highest viral titers and lowest viral RNA multiplication threshold. The plaque phenotypes also varied among the variants, in which the Beta and Omicron variants formed the largest and smallest plaques, respectively. All studied variants showed strong cytopathic effects after 48 h.p.i. The whole-genome sequencing highlighted cell-culture adaptation, where the Beta, Delta, and Omicron variants acquired mutations at the multibasic cleavage site after three cycles of passaging. The findings suggest a strong link between the replication rates and their respective transmissibility and pathogenicity. This is essential in predicting the impacts of the upcoming variants on the local and global populations and is useful in designing preventive measures to curb virus outbreaks. Full article

Highly Pathogenic Avian Influenza Viruses (HPAIVs) arise from low pathogenic precursors following spillover from wild waterfowl into poultry populations. The main virulence determinant of HPAIVs is the presence of a multi-basic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein. The MBCS allows for HA cleavage and, consequently, activation by ubiquitous proteases, which results in systemic dissemination in terrestrial poultry. Since 1959, 51 independent MBCS acquisition events have been documented, virtually all in HA from the H5 and H7 subtypes. In the present article, data from natural LPAIV to HPAIV conversions and experimental in vitro and in vivo studies were reviewed in order to compile recent advances in understanding HA cleavage efficiency, protease usage, and MBCS acquisition mechanisms. Finally, recent hypotheses that might explain the unique predisposition of the H5 and H7 HA sequences to obtain an MBCS in nature are discussed. Full article

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes. Full article