Despite the reduced fitness of the resistant H5N1 D1.1 variant, its emergence in the absence of viral selection pressure underscores the need for continued surveillance.
Abstract
ABSTRACT Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b, genotype D1.1, are responsible for widespread outbreaks in poultry and continue to cause sporadic, sometimes severe, human infections. Herein, we characterized a wild-type (WT) influenza A(H5N1) D1.1 isolate (BC-H5N1-WT) and its H275Y neuraminidase (NA) variant (BC-H5N1-H275Y), both of which emerged on farms in British Columbia, Canada, during the fall 2024 outbreak. In vitro analysis assessed replication kinetics in MDCK cells, with supernatants collected at different days post-infection (p.i.) and titrated by TCID50 and qRT-PCR. Neuraminidase inhibitor (NAI) susceptibility was determined by NA inhibition assays, whereas susceptibility to baloxavir acid (BXA) was evaluated by plaque reduction assay. In vivo virulence was evaluated in BALB/c mice infected with serial 10-fold dilutions of each virus to monitor weight loss and mortality. Viral titers in lungs, brain, nose, kidney, spleen, and heart were quantified at day 4 p.i. The BC-H5N1-WT virus was susceptible to the four antivirals tested, whereas BC-H5N1-H275Y displayed resistance to oseltamivir and peramivir but remained susceptible to zanamivir and BXA. The BC-H5N1-WT exhibited significantly higher viral replication titers than BC-H5N1-H275Y at all tested time points and showed larger plaque sizes. In mice, BC-H5N1-WT was more virulent with LD50 values of 1.78 × 103 PFUs compared to 8.71 × 104 PFUs for BC-H5N1-H275Y, and produced higher viral titers in lungs and other organs. Despite the reduced fitness of the resistant H5N1 D1.1 variant, its emergence in the absence of viral selection pressure underscores the need for continued surveillance.
Using the ferret model of influenza infection and transmission, it is demonstrated that the currently circulating B3.3.13 is already well adapted for mammalian infection and transmission whereas D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential.
Ahmed M. Elsayed, Ramya S. Barre, Mahmoud Bayoumi et al.· bioRxiv· 0 citations
Abstract Avian influenza A(H5N1) is a highly pathogenic virus causing significant out-breaks in birds and sporadic human infections since 1997. The virus is classified into various clades based on genetic characteristics, and recently, concerns have been raised over the risk ofclade 2.3.4.4b becoming widespread and infecting humans. Since 1997, more than 1,000 infections with highly pathogenic avian influenza A(H5N1) virus among humans have been reported globally. Given ongoing A(H5N1) outbreaks in animals, understanding the frequency of A(H5N 1) virus infections ‘among asymptomatic persons can inform public health risk assessments and infection prevention guidance. We performed a literature search focusing on reported infections amona humans with Highly Pathogenic Avian Influenza (HPAI). This study was conceived as a narrative review focusing on clade 2.3.4.4b H5N1, but was expanded to all reported H5Nx and other zoonotic subtypes (H5N6, H6N1, H7N9, H10N3) when clade-specific data were sparse. Although not a formal systematic review, the search, screening, and appraisal followed the PRISMA 2020 Checklist for search reporting where applicable.
P. Velikov, T. Valkov· Acta Medica Bulgarica· 0 citations
High pathogenicity avian influenza (HPAI) H5 clade 2.3.4.4b is the main driver of the ongoing unprecedented global panzootic. The recently emerged HPAI H5N1 clade 2.3.4.4b genotype EA-2024-DI.2.1 has become predominant in Europe, with migratory wild birds, particularly waterfowl, playing a major role in its dissemination. Egypt lies along major Afro-Eurasian migratory flyways, which have historically played an important role in the introduction of emerging H5Nx viruses into the country. In this study, targeted surveillance was conducted on 416 wild birds offered for sale in in live bird markets (LBMs) and roadside trading points in northern Egypt, mainly in Damietta and Port Said. Of these, 118 birds showing mild clinical signs were examined post-mortem and lung and tracheal tissues were collected, while oropharyngeal and cloacal swabs were collected from apparently healthy birds. Avian influenza virus was detected by RT-qPCR in 22 wild birds, all from tissue samples, whereas all swabs from apparently healthy birds were negative. Waterfowl accounted for 16 of the 22 positive birds (72.7%), with Eurasian teal showing the lowest Ct values (21-25). Phylogenetic and whole-genome analyses showed that the sequenced wild-bird viruses clustered within the recently emerged EA-2024-DI.2.1 sub-lineage and were closely related to contemporary European viruses. Compared with the EA-2021-AB genotype currently circulating in Egyptian poultry, the EA-2024-DI.2.1 viruses showed several HA amino acid differences, including A83D, L104M and T195A. These findings provide evidence for the introduction of EA-2024-DI.2.1 into Egypt through migratory wild birds and highlight the importance of continued genomic surveillance at the wild bird domestic poultry interface and antigenic evaluation against vaccines currently used in Egypt.
N. Hagag, Amany Adel, Mostafa R. Zaher et al.· bioRxiv· 0 citations
ABSTRACT Background H5Nx goose/Guangdong (Gs/GD) lineage highly pathogenic avian influenza (HPAI) viruses pose a significant public health threat due to their global spread, mutation accumulation, and expanding host range. The descendant clade 2.3.4.4b has been causing widespread infections in birds and increasing spillover events in mammals. Methods This report documents the first fatal case of highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b infection in a domestic cat in Italy, detected in early January 2025. The cat (CAT 1) resided on a backyard poultry farm experiencing a high pathogenicity avian influenza outbreak and succumbed rapidly following the onset of respiratory signs. A second exposed cat (CAT 2) developed clinical disease without fatal outcome. Comprehensive outbreak investigations were conducted, including pathological, serological, molecular analyses, and genomic characterization. Results Pathological examination of CAT 1 revealed acute necrotizing bronchointerstitial pneumonia, non‐suppurative meningoencephalitis, and disseminated foci of hepatic necrosis. Interestingly, the PB2‐E627K mutation associated with mammalian virus adaptation was observed in the feline viral isolate compared to avian isolates. Such polymerase complex mutations are key determinants of host range and increase pathogenicity in mammals. CAT 2 from the same farm tested negative for AIV genome detection but subsequently seroconverted for antibodies against NPA, H5, and N1. Conclusion Sharing these findings is crucial for surveillance aimed at enabling early identification of increased risks to human and animal health, preventing cross‐species viral transmission and mitigating the risk of potential spillover events.
G. D’Annunzio, Ana Moreno, Giovanni Tosi et al.· Influenza and Other Respirat...· 0 citations
Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus continues to spread globally and sporadically transmits from avian reservoirs to mammalian hosts. In May 2024, H5N1 infections in young goats and alpacas in the United States were reported. Nevertheless, the overall susceptibility of camelids to clade 2.3.4.4b virus remains unclear. We conducted a controlled experimental infection study in 6 alpacas, assessing clinical signs, viral shedding, tissue distribution, and serologic responses after intranasal inoculation with HPAI H5N1 genotype B3.13 virus. Observed illness was generally mild; body temperature increased slightly and food intake reduced for up to 3 days postinfection. We detected viral RNA in nasal swab samples and confirmed infectious HPAI H5N1 virus. Immunohistochemistry and RNA in situ hybridization detected virus only in the nasopharyngeal tonsil and nasal conchae at 4 days postinfection. Our findings suggest alpacas are susceptible to productive H5N1 infection, highlighting implications for livestock surveillance and biosecurity in regions with ongoing circulation.
Jacob Schön, A. Breithaupt, N. Halwe et al.· Emerging Infectious Diseases· 0 citations
ABSTRACT Background The high‐pathogenicity avian influenza (HPAI) viruses represent a main threat to animal and human health, poultry productivity, and ecosystems. The newly evolved A(H5N1) clade 2.3.4.4b has become a worldwide concern for its expanding multihost ecology, including poultry and a wide range of mammalian species. Methods Avian influenza virus (AIV) was isolated from an outbreak in commercial quail farms associated with severe respiratory signs and high mortality. Molecular characterization of the detected virus was performed through gene sequencing via whole‐genome sequencing technology and phylogenetic analysis. Results Genetic and phylogenetic analyses revealed that the hemagglutinin (HA) gene clustered closely with a recent HPAI A(H5N1) clade 2.3.4.4b strain from live bird markets (LBMs) in Bangladesh, with a most recent common ancestor dated to July 2023. The NA gene and four internal genes (PB1, NP, MP, NS) were derived from contemporary A(H5N1) viruses of ducks in LBMs, while the PB2 and PA segments exhibited similarity to low pathogenicity avian influenza (LPAI) viruses, suggesting reassortment events within avian hosts. The presence of a multibasic cleavage site (PLREKRRKR↓GLF) with avian‐specific receptor‐binding residues (Q224 and G226) and five conserved glycosylation motifs, consistent with high‐pathogenicity phenotypes and strong avian receptor affinity. The PB2 gene harbored a rare I147T substitution; however, key mammalian adaptation markers such as E627K and D701N were absent. Conclusion The reassortant A(H5N1) clade 2.3.4.4b virus with high pathogenicity and genetic links to LBM‐origin strains is now circulating in the commercial poultry population in Bangladesh, increasing the risk of viral adaptation, the potential for cross‐species transmission, and threatening the poultry sector and public health.
M. A. Samad, Md Rezaul Karim, Amelia Coggon et al.· Influenza and Other Respirat...· 0 citations
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