H5N1 clade 2.3.4.4b avian influenza viruses replicate in differentiated bovine airway epithelial cells cultured at air-liquid interface.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.

In Ovo Models to Predict Virulence of Highly Pathogenic Avian Influenza H5-Viruses for Chickens and Ducks.

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021-2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations.

During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health.

Antiviral Polymer Brushes by Visible-Light-Induced, Oxygen-Tolerant Covalent Surface Coating.

This work presents a novel route for creating metal-free antiviral coatings based on polymer brushes synthesized by surface-initiated photoinduced electron transfer-reversible addition-fragmentation chain transfer (SI-PET-RAFT) polymerization, applying eosin Y as a photocatalyst, water as a solvent, and visible light as a driving force. The polymer brushes were synthesized using N-[3-(decyldimethyl)-aminopropyl] methacrylamide bromide and carboxybetaine methacrylamide monomers. The chemical composition, thickness, roughness, and wettability of the resulting polymer brush coatings were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), water contact angle measurements, and ellipsometry. The antiviral properties of coatings were investigated by exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian influenza viruses, with further measurement of residual viable viral particles. The best performance was obtained with Cu surfaces, with a ca. 20-fold reduction of SARS-Cov-2 and a 50-fold reduction in avian influenza. On the polymer brush-modified surfaces, the number of viable virus particles decreased by about 5-6 times faster for avian flu and about 2-3 times faster for SARS-CoV-2, all compared to unmodified silicon surfaces. Interestingly, no significant differences were obtained between quaternary ammonium brushes and zwitterionic brushes.

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 viruses.

Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed that chickens are efficiently infected by (fecal) contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

The development of a multiplex serological assay for avian influenza based on Luminex technology.

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.

Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy.

After avian influenza (AI) vaccination, hens will produce progeny chickens with maternally derived AI-specific antibodies. In the present study we examined the effect of maternal immunity in young chickens on the protection against highly pathogenic AI H5N1 virus infection and on the effectiveness of AI vaccination. The mean haemagglutination inhibition antibody titre in sera of 14-day-old progeny chickens was approximately eight-fold lower than the mean titre in sera of vaccinated hens. After H5N1 infection at the age of 14 days, chickens with maternal antibody titres lived a few days longer than control chickens. However, only a low proportion of chickens with maternal immunity survived challenge with H5N1. In most progeny chickens with maternal immunity, high virus titres (>10(4) median embryo infective dose) were present in the trachea during the first 4 days after H5N1 infection. In the cloaca, only low virus titres were present in most chickens. In 14-day-old progeny chickens with maternal immunity, the induction of antibody titres by vaccination was severely inhibited, with only a few chickens showing responses similar to the control chickens. It is concluded that high maternal antibody titres are required for clinical protection and reduction of virus titres after infection of chickens, whereas low antibody titres already interfere with vaccine efficacy.

Quantification of infectious bursal disease viral proteins 2 and 3 in inactivated vaccines as an indicator of serological response and measure of potency.

Viral protein 2 and viral protein 3 (VP2 and VP3) were quantified in a series of inactivated infectious bursal disease oil emulsion vaccines using enzyme-linked immunosorbent assay, and the dependence of the serological response on vaccine antigen content was studied. Large differences in antigen content, up to 50-fold, were found between vaccines. Neutralizing antibody titres at 3 to 6 weeks after vaccination varied from 3 log2 to 16 log2. None of the vaccines induced an antibody titre equal to that of the reference serum used as an indicator of sufficient potency in the European Pharmacopoeia. Neutralizing antibody titres after vaccination correlated highly with the VP2 content of the vaccines. A significant correlation was also found between the VP3 content and the antibody response. Our data illustrate that the antigen content of inactivated infectious bursal disease vaccines is a reliable indicator of the protective serological response after vaccination, and consequently could be used as a measure of vaccine potency. This holds true for both VP2, the antigen that induces neutralizing antibodies, as well as for VP3, which does not induce neutralizing antibodies.