Avian H5 Research Articles

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge.

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity-either from LAIV vaccination and/or from Cal09 infection-showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

The mammary glands of cows abundantly display receptors for circulating avian H5 viruses.

Influenza A viruses (IAVs) from the H5N1 2.3.4.4b clade are circulating in dairy farms in the USA.; ruminants were presumed not to be hosts for IAVs. Previously, IAV-positive mammalian species were hunters and scavengers, possibly getting infected while feeding on infected birds. It is now recognized that H5N1 viruses that circulate in US dairy cattle transmit through a mammary gland route, in contrast to transmission by aerosols via the respiratory tract. The sialome in the cow mammary and respiratory tract is so far solely defined using plant lectins. Here, we used recombinant HA proteins representing current circulating and classical H5 viruses to determine the distribution of IAV receptors in the respiratory and mammary tract tissues of cows. We complemented our study by mapping the glycan distribution of the upper and lower respiratory tracts of horses and pigs. Most of the sialome of the cow respiratory tract is lined with sialic acid modifications, such as N-glycolyl and O-acetyl, which are not bound by IAV. Interestingly, the H5 protein representing the cow isolates is bound significantly in the mammary gland, whereas classical H5 proteins failed to do so. Furthermore, whereas the 9-O-acetyl modification is prominent in all tissues tested, the 5-N-glycolyl modification is not, resulting in the display of receptors for avian IAV hemagglutinins. This could explain the high levels of virus found in these tissues and milk, adding supporting data to this virus transmission route.IMPORTANCEH5N1 influenza viruses, which usually affect birds, have been found on dairy farms in the USA. Surprisingly, these viruses are spreading among dairy cows, and there is a possibility that they do not spread through the air but through their milk glands. To understand this better, we studied how the virus attaches to tissues in the cow's respiratory tract and mammary glands using specific viral proteins. We found that the cow-associated virus binds strongly to the mammary glands, unlike older versions infecting birds. This might explain why the virus is found in cow's milk, suggesting a new way the virus could be spreading.

Pichia pastoris an Ideal Host for the Production of Recombinant Influenza Vaccines

Pichia pastoris is a methylotrophic yeast with remarkable characteristics such as lacking endotoxin, producing high amounts of recombinant protein, performing post-translational modifications, and so on. Influenza A virus, a member of the Orthomyxoviridae family, is the cause of avian influenza. Three avian influenza virus subtypes, H5, H7 and H9, are commercially and physiologically significant in the poultry industry. Some researchers considered influenza to be the next pandemic disease. Nowadays, researchers have paid attention to producing novel and effective recombinant vaccines, especially in the poultry industry. Due to the advantages of P. pastoris yeast, it can be used as an ideal expression system for producing subunit vaccines. Although several studies have been conducted in this field, there is no comprehensive review of using P. pastoris to produce recombinant influenza vaccines. This review explains the different strains, phenotypes, and advantages of this yeast and then the production of recombinant influenza vaccines using this expression system is discussed in detail.

A case report of human infection with avian influenza H10N3 with a complex respiratory disease history

BackgroundOn March 16th 2024, the first case of Human infection with avian influenza H10N3 since the end of the global COVID-19 Pandemic was reported in Kunming, China. To enhance comprehension of the source of infection and risk factors of the H10N3 virus infection, this case report summarizes the clinical features, epidemiological investigation, and laboratory test results. Provides recommendations for the prevention and control of Human infection with avian influenza H10N3.Case presentationA 51-year-old male with a history of COVID-19 infection and a smoking habit of 30 years, worked in livestock breeding and was exposed to sick and dead poultry before falling ill with fever and chills on 28th February 2024. A week later, he was diagnosed with severe pneumonia, influenza, and respiratory failure by the Third People's Hospital of Kunming(KM-TPH). He was discharged on 17th April and none of his 6 close contacts showed any symptoms of illness. Environmental samples taken from the epidemic spot revealed that peacock feces tested positive for avian influenza sub-type H9 and waterfowl specimens showed positive results for avian influenza sub-type H5. Gene sequencing conducted on positive specimens from the patient's respiratory tract by the Chinese Centre for Disease Control and Prevention (CCDC) showed a high degree of similarity (98.6–99.5%) with the strain responsible for the second global case of human infected with H10N3 (reported from Zhejiang, China 2022).ConclusionsAccording to the available epidemiological information, there is limited evidence to suggest that H10N3 viruses are excessively lethal. However, adaptive site mutations have been observed in the H10N3 isoform of mammals. While it is unlikely that the H10N3 virus will spread among humans, the possibility of additional cases cannot be entirely ruled out. Symptoms of human infection with H10N3 avian influenza are similar to those of common respiratory infections, which may result in them being overlooked during initial clinical consultations. Therefore, it is essential to improve surveillance of the H10 sub-type of avian influenza and to increase the awareness of hospital-related workers of cases of pneumonia of unknown origin.

Collaborative wildlife disease outbreak investigation and response at Bells Swamp Victoria, February 2023.

In February 2023, a report of morbidity and mortality in waterbirds triggered a collaborative regional wildlife disease outbreak investigation and response, led by Parks Victoria. Triage, rehabilitation and diagnosis of sick and dead birds were undertaken by Zoos Victoria (ZV), Agriculture Victoria, Vets for Compassion, Wildlife Victoria and Melbourne Veterinary School (MVS). The field response focused on collection of sick and dead birds for wildlife welfare, for diagnosis, and to reduce environmental contamination. Botulism was suspected, based on clinical signs and lack of significant gross pathology, and this diagnosis was confirmed by PCR testing. Low pathogenicity avian influenza (LPAI) viruses non H5 or H7 were detected in two birds and ruled out in all in others tested. These incidental, non-clinical LPAI detections are considered part of the natural wild bird virus community in Australia. A number of elements contributed to the collaborative effort. Regional individuals had the necessary connections for reporting, collecting and transporting birds. There was rapid determination by the Victorian Department of Energy, Environment and Climate Action (DEECA) that Parks Victoria, as the land managers, should lead the response. Zoos Victoria provided capacity and expertise in wildlife triage and rehabilitation, and Agriculture Victoria, ZV and MVS were responsible for veterinary management of the response and diagnosis. Field investigation and response were conducted by Parks Victoria, Agriculture Victoria, MVS and veterinary teams from Vets for Compassion and Wildlife Victoria. Wildlife Health Australia (WHA) provided guidance and information, approved National Significant Disease Investigation Program funding and captured the event in the national wildlife health information database. Communication and media were important for community understanding of the event.

MHC class II proteins mediate sialic acid independent entry of human and avian H2N2 influenza A viruses.

Influenza A viruses (IAV) pose substantial burden on human and animal health. Avian, swine and human IAV bind sialic acid on host glycans as receptor, whereas some bat IAV require MHC class II complexes for cell entry. It is unknown how this difference evolved and whether dual receptor specificity is possible. Here we show that human H2N2 IAV and related avian H2N2 possess dual receptor specificity in cell lines and primary human airway cultures. Using sialylation-deficient cells, we reveal that entry via MHC class II is independent of sialic acid. We find that MHC class II from humans, pigs, ducks, swans and chickens but not bats can mediate H2 IAV entry and that this is conserved in Eurasian avian H2. Our results demonstrate that IAV can possess dual receptor specificity for sialic acid and MHC class II, and suggest a role for MHC class II-dependent entry in zoonotic IAV infections.

Effects of Allicin on the Immune Performance and Expression of Immune-related Genes in Muchuan Black-bone Chickens

Background: In response to China’s ban on medicinal feed additives with growth-promoting functions in livestock and poultry feed, there is an urgent need in modern animal husbandry for alternatives to antibiotics to sustain livestock health and improve feed conversion efficiency. Allicin, known for its safety, efficiency, lack of drug resistance and absence of residue, regulates the intestinal flora structure; inhibits the growth of harmful bacteria; improves intestinal health, immunity and stress resistance; and improves animal production. Methods: To investigate the impact of allicin on immune performance and immune-related gene expression in Muchuan black-bone chickens, diets were supplemented with varying allicin levels (50, 100, 200 mg/kg), designated as test groups A, B and C, respectively. Initially, growth performance was assessed. Subsequently, immunoglobulins were measured via enzyme-linked immunosorbent assay (ELISA) and colorimetry; immune titers for avian influenza and Newcastle disease were evaluated using hemagglutination and hemagglutination inhibition tests and the expression of immune genes was analyzed via real-time quantitative PCR. Finally, serum biochemical indices in each group were determined. Result: Compared to the control group, allicin supplementation resulted in increased average fasting body weight in the experimental group and significantly (P less than 0.05) higher thymus and musogastric indices in groups A and C, respectively. Serum levels of immunoglobulin M in groups A and B and immunoglobulin G levels were significantly higher in all experimental groups than in controls. Antibody titers against avian influenza H5 and H7 strains were higher in all test groups, with Newcastle disease antibody titers in groups B and C significantly higher than those in controls. The mRNA expression levels of IFN-ã were lower across all experimental groups, whereas the expression levels of TLR3 in groups B and C and TLR15 in group B were higher. Serum levels of triglycerides in group A were significantly higher and blood urea nitrogen levels were significantly lower in groups B and C than in controls. These findings indicate that dietary allicin (50-200 mg/kg) significantly improves growth performance, immunity and protein and lipid metabolism in Muchuan black-bone chickens, offering valuable data for the development and use of allicin as an antibiotic alternative.

Does pasteurization inactivate bird flu virus in milk?

ABSTRACT Recently, an outbreak of highly pathogenic avian influenza A (H5N1), which carries the clade 2.3.4.4b hemagglutinin (HA) gene and has been prevalent among North American bird populations since the winter of 2021, was reported in dairy cows in the United States. As of 24 May 2024, the virus has affected 63 dairy herds across nine states and has resulted in two human infections. The virus causes unusual symptoms in dairy cows, including an unexpected drop in milk production, and thick colostrum-like milk. Notably, The US Food and Drug Administration reported that around 20% of tested retail milk samples contained H5N1 viruses, with a higher percentage of positive results from regions with infected cattle herds. Data are scant regarding how effectively pasteurization inactivates the H5N1 virus in milk. Therefore, in this study, we evaluated the thermal stability of the H5 clade 2.3.4.4b viruses, along with one human H3N2 virus and other influenza subtype viruses, including H1, H3, H7, H9, and H10 subtype viruses. We also assessed the effectiveness of pasteurization in inactivating these viruses. We found that the avian H3 virus exhibits the highest thermal stability, whereas the H5N1 viruses that belong to clade 2.3.4.4b display moderate thermal stability. Importantly, our data provide direct evidence that the standard pasteurization methods used by dairy companies are effective in inactivating all tested subtypes of influenza viruses in raw milk. Our findings indicate that thermally pasteurized milk products do not pose a safety risk to consumers.

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9

BackgroundAvian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA.ResultsThe results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%.ConclusionsRT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

Generation and characterization of a nanobody against the avian influenza virus H7 subtype

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-DiGS, Nb-Z77-TriGS, Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-DiGS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-DiGS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 μg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection.

Avian influenza overview December 2023-March 2024.

Between 2 December 2023 and 15 March 2024, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (227) and wild (414) birds across 26 countries in Europe. Compared to previous years, although still widespread, the overall number of HPAI virus detections in birds was significantly lower, among other reasons, possibly due to some level of flock immunity in previously affected wild bird species, resulting in reduced contamination of the environment, and a different composition of circulating A(H5N1) genotypes. Most HPAI outbreaks reported in poultry were primary outbreaks following the introduction of the virus by wild birds. Outside Europe, the majority of outbreaks in poultry were still clustered in North America, while the spread of A(H5) to more naïve wild bird populations on mainland Antarctica is of particular concern. For mammals, A(H5N5) was reported for the first time in Europe, while goat kids in the United States of America represented the first natural A(H5N1) infection in ruminants. Since the last report and as of 12 March 2024, five human avian influenza A(H5N1) infections, including one death, three of which were clade 2.3.2.1c viruses, have been reported by Cambodia. China has reported two human infections, including one fatal case, with avian influenza A(H5N6), four human infections with avian influenza A(H9N2) and one fatal case with co-infection of seasonal influenza A(H3N2) and avian influenza A(H10N5). The latter case was the first documented human infection with avian influenza A(H10N5). Human infections with avian influenza remain rare and no sustained human-to-human infection has been observed. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for those occupationally or otherwise exposed to infected animals.

Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses.

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.

Avian influenza overview September-December 2023.

Between 2 September and 1 December 2023, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (88) and wild (175) birds across 23 countries in Europe. Compared to previous years, the increase in the number of HPAI virus detections in waterfowl has been delayed, possibly due to a later start of the autumn migration of several wild bird species. Common cranes were the most frequently affected species during this reporting period with mortality events being described in several European countries. Most HPAI outbreaks reported in poultry were primary outbreaks following the introduction of the virus by wild birds, with the exception of Hungary, where two clusters involving secondary spread occurred. HPAI viruses identified in Europe belonged to eleven different genotypes, seven of which were new. With regard to mammals, the serological survey conducted in all fur farms in Finland revealed 29 additional serologically positive farms during this reporting period. Wild mammals continued to be affected mostly in the Americas, from where further spread into wild birds and mammals in the Antarctic region was described for the first time. Since the last report and as of 1 December 2023, three fatal and one severe human A(H5N1) infection with clade 2.3.2.1c viruses have been reported by Cambodia, and one A(H9N2) infection was reported from China. No human infections related to the avian influenza detections in animals in fur farms in Finland have been reported, and human infections with avian influenza remain a rare event. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated); this assessment covers different situations that depend on the level of exposure.

Avian influenza overview June-September 2023.

Between 24 June and 1 September 2023, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (25) and wild (482) birds across 21 countries in Europe. Most of these outbreaks appeared to be clustered along coastlines with only few HPAI virus detections inland. In poultry, all HPAI outbreaks were primary and sporadic with most of them occurring in the United Kingdom. In wild birds, colony-breeding seabirds continued to be most heavily affected, but an increasing number of HPAI virus detections in waterfowl is expected in the coming weeks. The current epidemic in wild birds has already surpassed the one of the previous epidemiological year in terms of total number of HPAI virus detections. As regards mammals, A(H5N1) virus was identified in 26 fur animal farms in Finland. Affected species included American mink, red and Arctic fox, and common raccoon dog. The most likely source of introduction was contact with gulls. Wild mammals continued to be affected worldwide, mostly red foxes and different seal species. Since the last report and as of 28 September 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans have been reported by the United Kingdom, and three human infections with A(H5N6) and two with A(H9N2) were reported from China, respectively. No human infection related to the avian influenza detections in animals on fur farms in Finland or in cats in Poland have been reported, and human infections with avian influenza remain a rare event. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated); this assessment covers different situations that depend on the level of exposure.

Avian influenza overview April - June 2023.

Between 29 April and 23 June 2023, highly pathogenic avian influenza (HPAI) A(H5N1) virus (clade 2.3.4.4b) outbreaks were reported in domestic (98) and wild (634) birds across 25 countries in Europe. A cluster of outbreaks in mulard ducks for foie gras production was concentrated in Southwest France, whereas the overall A(H5N1) situation in poultry in Europe and worldwide has eased. In wild birds, black-headed gulls and several new seabird species, mostly gulls and terns (e.g. sandwich terns), were heavily affected, with increased mortality being observed in both adults and juveniles after hatching. Compared to the same period last year, dead seabirds have been increasingly found inland and not only along European coastlines. As regards mammals, A(H5N1) virus was identified in 24 domestic cats and one caracal in Poland between 10 and 30 June 2023. Affected animals showed neurological and respiratory signs, sometimes mortality, and were widely scattered across nine voivodeships in the country. All cases are genetically closely related and identified viruses cluster with viruses detected in poultry (since October 2022, but now only sporadic) and wild birds (December 2022-January 2023) in the past. Uncertainties still exist around their possible source of infection, with no feline-to-feline or feline-to-human transmission reported so far. Since 10 May 2023 and as of 4 July 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans were reported from the United Kingdom, and two A(H9N2) and one A(H5N6) human infections in China. In addition, one person infected with A(H3N8) in China has died. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA, low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated).

A Novel Triple Reassortment H3N8 Avian Influenza Virus: Characteristics, Pathogenicity, and Transmissibility

An increasing number of new subtypes of avian influenza viruses (AIVs) are reported to be infecting humans, including H3N2, H5N1, H7N9, H10N8, and the recently emerged H3N8 virus in China in 2022. However, the genetic and biological properties of the currently prevalent H3N8 AIVs are not yet fully understood. This study reports the isolation of a novel triple reassortment H3N8 virus (GD-H3N8) from chicken flocks in Guangdong province, China, in 2022. The GD-H3N8 virus contains the Eurasian avian duck-origin H3 gene, the North American avian N8 gene, and dynamic internal genes of the H9N2 virus, and shows high homology with human H3N8 strains. The GD-H3N8 isolate has multiple mammalian adaptive mutations associated with receptor binding and virulence. Growth kinetics assays demonstrate that the GD-H3N8 isolate is capable of efficient replication in avian, mammalian, and human cells in vitro. In vivo, the GD-H3N8 isolate can replicate efficiently in mice without preadaptation, in addition to establishing systemic infection and transmission by direct contact in chickens. These findings underscore the need for continued surveillance of H3N8 viruses to identify circulating strains that may potentially threaten human health.

Characterization of an Emergent Chicken H3N8 Influenza Virus in Southern China: a Potential Threat to Public Health.

Although influenza A viruses of several subtypes have occasionally infected humans, to date only those of the H1, H2, and H3 subtypes have led to pandemics and become established in humans. The detection of two human infections by avian H3N8 viruses in April and May of 2022 raised pandemic concerns. Recent studies have shown the H3N8 viruses were introduced into humans from poultry, although their genesis, prevalence, and transmissibility in mammals have not been fully elucidated. Findings generated from our systematic influenza surveillance showed that this H3N8 influenza virus was first detected in chickens in July 2021 and then disseminated and became established in chickens over wider regions of China. Phylogenetic analyses revealed that the H3 HA and N8 NA were derived from avian viruses prevalent in domestic ducks in the Guangxi-Guangdong region, while all internal genes were from enzootic poultry H9N2 viruses. The novel H3N8 viruses form independent lineages in the glycoprotein gene trees, but their internal genes are mixed with those of H9N2 viruses, indicating continuous gene exchange among these viruses. Experimental infection of ferrets with three chicken H3N8 viruses showed transmission through direct contact and inefficient transmission by airborne exposure. Examination of contemporary human sera detected only very limited antibody cross-reaction to these viruses. The continuing evolution of these viruses in poultry could pose an ongoing pandemic threat. IMPORTANCE A novel H3N8 virus with demonstrated zoonotic potential has emerged and disseminated in chickens in China. It was generated by reassortment between avian H3 and N8 virus(es) and long-term enzootic H9N2 viruses present in southern China. This H3N8 virus has maintained independent H3 and N8 gene lineages but continues to exchange internal genes with other H9N2 viruses to form novel variants. Our experimental studies showed that these H3N8 viruses were transmissible in ferrets, and serological data suggest that the human population lacks effective immunological protection against it. With its wide geographical distribution and continuing evolution in chickens, other spillovers to humans can be expected and might lead to more efficient transmission in humans.

T-705-Derived Prodrugs Show High Antiviral Efficacies against a Broad Range of Influenza A Viruses with Synergistic Effects When Combined with Oseltamivir.

Emerging influenza A viruses (IAV) bear the potential to cause pandemics with unpredictable consequences for global human health. In particular, the WHO has declared avian H5 and H7 subtypes as high-risk candidates, and continuous surveillance of these viruses as well as the development of novel, broadly acting antivirals, are key for pandemic preparedness. In this study, we sought to design T-705 (Favipiravir) related inhibitors that target the RNA-dependent RNA polymerase and evaluate their antiviral efficacies against a broad range of IAVs. Therefore, we synthesized a library of derivatives of T-705 ribonucleoside analogues (called T-1106 pronucleotides) and tested their ability to inhibit both seasonal and highly pathogenic avian influenza viruses in vitro. We further showed that diphosphate (DP) prodrugs of T-1106 are potent inhibitors of H1N1, H3N2, H5N1, and H7N9 IAV replication. Importantly, in comparison to T-705, these DP derivatives achieved 5- to 10-fold higher antiviral activity and were non-cytotoxic at the therapeutically active concentrations. Moreover, our lead DP prodrug candidate showed drug synergy with the neuraminidase inhibitor oseltamivir, thus opening up another avenue for combinational antiviral therapy against IAV infections. Our findings may serve as a basis for further pre-clinical development of T-1106 prodrugs as an effective countermeasure against emerging IAVs with pandemic potential.

Avian influenza overview March – April 2023

Between 2 March and 28 April 2023, highly pathogenic avian influenza (HPAI) A(H5Nx) virus, clade 2.3.4.4b, outbreaks were reported in domestic (106) and wild (610) birds across 24 countries in Europe. Poultry outbreaks occurred less frequently compared to the previous reporting period and compared to spring 2022. Most of these outbreaks were classified as primary outbreaks without secondary spread and some of them associated with atypical disease presentation, in particular low mortality. In wild birds, black-headed gulls continued to be heavily affected, while also other threatened wild bird species, such as the peregrine falcon, showed increased mortality. The ongoing epidemic in black-headed gulls, many of which breed inland, may increase the risk for poultry, especially in July-August, when first-year birds disperse from the breeding colonies. HPAI A(H5N1) virus also continued to expand in the Americas, including in mammalian species, and is expected to reach the Antarctic in the near future. HPAI virus infections were detected in six mammal species, particularly in marine mammals and mustelids, for the first time, while the viruses currently circulating in Europe retain a preferential binding for avian-like receptors. Since 13 March 2022 and as of 10 May 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans were reported from China (1), and Chile (1), as well as three A(H9N2) and one A(H3N8) human infections in China. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA, and low to moderate for occupationally or otherwise exposed people.

Antibody prevalence to avian influenza virus subtypes H5, H7 and H9 in falcons, captive and wild birds, United Arab Emirates, 2003-2006.

Avian influenza viruses (AIV) may cause enormous economic losses in the poultry industry and sporadically severe disease in humans. Falconry is a tradition of great importance in the Arabian Peninsula. Falcons may catch AIV through contact with infected quarry species. Falcons together with other bird species are the focus of this seroprevalence study, carried out on sera collected in the United Arab Emirates (UAE). AIV with the haemagglutinin subtypes H5, H7 and possibly H9 may infect humans. We investigated the antibody prevalence to these subtypes in falcons and other birds by haemagglutination inhibition test. 617 sera of falcons and 429 sera of 46 wild/captive bird species were tested. From the falcons, only one was positive for H5 antibodies (0.2%), none contained antibodies to H7, but 78 had antibodies to H9 (13.2%). Regarding other birds, eight were positive for antibodies to H5 (2.1%), none had antibodies to H7, but 55 sera from 17 species contained antibodies to H9 (14.4%). In contrast to H5 and H7 infections, H9N2 is widespread worldwide. Its ability to reassort, thereby creating possibly pathogenic strains for humans, should remind us of the potential risk that close contact with birds entails.