Search

The H9N2 subtype of avian influenza virus (AIV) and infectious bronchitis virus (IBV) are important avian viruses that cause respiratory symptoms in poultry, and can form mixed infections. In this study, primers and probes were designed based on the HA gene of H9N2 and the 5′ noncoding region of IBV, respectively, and a fluorescent quantitative RT–PCR assay was established for simultaneous detection of these two pathogens. The reaction system and conditions were optimized. The method only detected AIV subtype H9N2 and IBV and no other viruses, confirming its high specificity. The assay detected 13.5 copies/μL and 1.66 copies/μL of H9N2 and IBV in clinical samples, respectively. The coefficients of variation for intra- and interassay repeatability were < 3%. The established method was used to analyze 254 clinical samples (oropharyngeal and cloacal swabs) from Hubei Province, China; 98.82% were positive for both pathogens. In summary, a duplex fluorescent quantitative RT–PCR method capable of simultaneously detecting AIV subtype H9N2 and IBV was established. It is specific, sensitive, and reproducible, and can be used for diagnosis of a variety of clinical samples. It provides a technological means for the rapid and simultaneous detection of both pathogens, and thus can facilitate clinical diagnosis and epidemiological investigations. [ABSTRACT FROM AUTHOR]

Rapid and accurate detection of H5Nx avian influenza viruses is critical for effective surveillance and control measures. Currently, RT-qPCR with spin column RNA extraction is the gold standard for HPAIV surveillance, but its long reaction time and need for specialized equipment limit its effectiveness for rapid response. In this study, we introduce a centrifuge-free, rapid detection method for on-site detection of H5Nx viruses that combines magnetic bead-based ribonucleoprotein (RNP) purification and concentration with a CRISPR-Cas12a system that is independent of the protospacer adjacent motif (PAM) sequence. Our approach employs anti-NP monoclonal antibodies for the targeted isolation of RNA bound to RNPs, facilitating a quick and specific RNA extraction process that negates the need for centrifugation. Additionally, by denaturing the RT-RPA amplicon using 60% DMSO, we activate the trans-ssDNA cleavage activity of the Cas12a protein without the need for a specific PAM (5'-TTTV-3′) sequence. This strategy increases flexibility in CRISPR RNA design, providing a significant advantage when targeting genes with high variability. We validated the efficacy of our magnetic RNP purification and concentration method in combined with an RT-RPA/PAM-independent Cas12a assay for detecting the H5 gene. The assay achieved a sensitivity threshold of 101 EID50 with fluorescent detection and 102 EID50 using lateral flow strips. It also exhibited high specificity, yielding positive results solely for H5Nx viruses among various influenza A virus subtypes. Furthermore, in clinical samples, the assay demonstrated 80% sensitivity and 100% specificity. These results highlight the advantages of using NP-specific antibodies for RNP purification and CRISPR-Cas12a with viral gene-specific crRNA to achieve exceptional diagnostic specificity. [ABSTRACT FROM AUTHOR]

H4Nx avian influenza viruses (AIVs) have been isolated from wild birds and poultry and can also cross the species barrier to infect mammals (pigs and muskrats). The widespread presence of these viruses in wild birds and poultry and their ability to be transmitted interspecies make them an undeniable hazard to the poultry farming industry. In the present study, we collected fecal and swab samples from wild birds and poultry in Guangdong Province from January 2019 to March 2024, and various subtypes of AIVs were isolated, including 19 strains of H4 subtype AIVs. Further analysis was conducted on the internal genes of the 19 strains. These strains clustered together with high homology to highly pathogenic avian influenza virus (HPAIV), suggesting that H4Nx AIV may be reassorted from HPAIV. Two H4N8 strains are phylogenetically related to the porcine H4N8 AIV. Molecular characterization revealed that all viruses in this study were less pathogenic but had potential mammalian-adapted mutations. The transmission dynamics of H4Nx AIVs revealed that Europe and Asia, especially the Netherlands and Bangladesh, may be the centers of transmission. This may be linked to the migration of wild birds. The high migration rates from Russia to the Netherlands and from Russia to Bangladesh may also play a role. Therefore, continuous and systematic monitoring of wild birds to clarify the spatial and temporal distribution and prevalence of influenza viruses in wild birds is significant for early warning of avian influenza outbreaks in poultry and for risk assessment for public health and safety. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) are highly contagious pathogens that cause zoonotic disease with limited availability of antiviral therapies, presenting ongoing challenges to both public health and the livestock industry. Unveiling host proteins that are crucial to the IAV life cycle can help clarify mechanisms of viral replication and identify potential targets for developing alternative host-directed therapies. Using a four-dimensional (4D), label-free methodology coupled with bioinformatics analysis, we analyzed the expression patterns of cellular proteins that changed following H9N2 virus infection. Compared to the control group, the H9N2 infected group displayed 732 differentially expressed proteins (DEPs), with 298 proteins showing upregulation and 434 proteins showing downregulation. Gene Ontology (GO) functional analysis showed that DEPs were catalog in 11 biological processes, three cellular components, and eight molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEPs were involved in processes including cytokine signaling pathways induced by virus infection and protein digestion and absorption. Proteins including TP53, DDX58, and STAT3 were among the top hub proteins in the protein–protein interaction (PPI) analysis, suggesting that these signaling cascades could be essential for the propagation of IAVs. Furthermore, the host protein SNAPIN was chosen to ascertain the accuracy of expression changes identified through a proteomic analysis. The results indicated that SNAPIN was downregulated following infection with IAVs both in vitro and in vivo, which is consistent with the proteomics results, suggesting that SNAPIN may serve as a key regulatory factor in the viral life cycle of IAVs. Our research delineates an extensive interaction map of IAV infection within the A549 cells, facilitating the discovery of pivotal proteins that contribute to the virus's propagation, potentially offering target candidates to screen for antiviral therapeutics. [ABSTRACT FROM AUTHOR]

The H3 subtype of avian influenza virus (AIV) stands out as one of the most prevalent subtypes, posing a significant threat to public health. In this study, a novel triple-reassortant H3N3 AIV designated A/chicken/China/16/2023 (H3N3), was isolated from a sick chicken in northern China. The complete genome of the isolate was determined using next-generation sequencing, and the AIV-like particles were confirmed via transmission electron microscopy. Subsequent phylogenetic analyses revealed that HA and NA genes of the H3N3 isolate clustered within the Eurasian lineage of AIVs, exhibiting the closest genetic relationship with other H3N3 AIVs identified in China during 2023. Interestingly, the HA and NA genes of the nove H3N3 isolate were originated from H3N8 and H10N3 AIVs, respectively, and the six internal genes originated from prevalent H9N2 AIVs. These findings indicated the novel H3N3 isolate possesses a complex genetic constellation, likely arising from multiple reassortment events involving H3N8, H9N2, and H10N3 subtype influenza viruses. Additionally, the presence of Q226 and T228 in the HA protein suggests the H3N3 virus preferentially binds to α-2,3-linked sialic acid receptors. The HA cleavage site motif (PEKQTR/GIF) and the absence of E627K and D701N mutations in PB2 protein classify the virus as a characteristic low pathogenicity AIV. However, several mutations in internal genes raise concerns about potential increases in viral resistance, virulence, and transmission in mammalian hosts. Overall, this study provides valuable insights into the molecular and genetic characterization of the emerging triple-reassortant H3N3 AIVs, and continued surveillance of domestic poultry is essential for monitoring the H3N3 subtype evolution and potential spread. [ABSTRACT FROM AUTHOR]

Objective: Avian influenza virus (AIV) infections first affect the respiratory tract of chickens. The epithelial cells activate the host immune system, which leads to the induction of immune-related genes and the production of antiviral molecules against external environmental pathogens. In this study, we used chicken tracheal epithelial cells (TECs) in vitro model to investigate the immune response of the chicken respiratory tract against avian respiratory virus infections. Methods: Eighteen-day-old embryonic chicken eggs were used to culture the primary chicken TECs. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) analysis of epithelial cell-specific gene makers were performed to confirm the characteristics, morphology, and growth pattern of primary cultured chicken TECs. Moreover, to investigate the cellular immune response to AIV infection or polyinosinic-polycytidylic acid (poly [I:C]) treatment, the TECs were infected with the H5N1 virus or poly (I:C). Then, immune responses were validated by RT-qPCR and western blotting. Results: The TECs exhibited polygonal morphology and formed colony-type cell clusters. The RT-qPCR results showed that H5N1 infection induced a significant expression of antiviral genes in TECs. We found that TECs treated with poly (I:C) and exposed to AIV infection-mediated activation of signaling pathways, leading to the production of antiviral molecules (e.g., pro-inflammatory cytokines and chemokines), were damaged due to the loss of junction proteins. We observed the activation of the nuclear factor kappa B and mitogen-activated protein kinase (MAPK) pathways, which are involved in inflammatory response by modulating the release of pro-inflammatory cytokines and chemokines in TECs treated with poly (I:C) and pathway inhibitors. Furthermore, our findings indicated that poly (I:C) treatment compromises the epithelial cell barrier by affecting junction proteins in the cell membrane. Conclusion: Our study highlights the utility of in vitro TEC models for unraveling the mechanisms of viral infection and understanding host immune responses in the chicken respiratory tract. [ABSTRACT FROM AUTHOR]

Human cases of avian influenza A(H5N2) and A(H5N1) viruses associated with outbreaks in birds and mammals are increasing globally, raising concerns about the possibility of a future avian influenza pandemic. We conducted a systematic review examining 30 reported cases of avian influenza in pregnant women. We found high mortality rates for mothers (90.0%, 27/30) and their babies (86.7%, 26/30) when women were infected with avian influenza virus during pregnancy. Despite being a high-risk population and having worse health outcomes across multiple pandemics, pregnant women are often excluded from vaccine trials. However, as the risk for a new pandemic increases and human vaccines against avian influenza are developed, early inclusion of pregnant women in clinical trials can inform the risk–benefit analysis for both the mother and their newborn infant. Early inclusion of pregnant women in public health vaccination programs is vital for protecting this high-risk population.

Testing of ducks in Tennessee, United States, before introduction of highly pathogenic influenza A(H5N1) virus demonstrated a high prevalence of antibodies to influenza A virus but very low prevalence of antibodies to H5 (25%) or H5 and N1 (13%) subtypes. Antibody prevalence increased after H5N1 introduction.

Background and Aim: Potassium peroxymonosulfate (PPMS) is a broad-spectrum disinfectant that oxidizes viral protein capsids. The effectiveness of PPMS in killing viruses depends on several factors, including its concentration, contact time, and present of organic materials. This study evaluated the efficacy of PPMS in an aqueous phase. It also applied PPMS to artificially avian influenza virus (AIV)-contaminated carrier surfaces and clothes and compared its effectiveness with that of sodium dichloroisocyanurate (NaDCC) and quaternary ammonium compounds (QAC). Materials and Methods: Four PPMS concentrations (1×, 0.5×, 0.25×, and 0.125×), were evaluated for their virucidal efficacy against Newcastle disease virus (NDV) and AIV in an aqueous phase. The evaluation included testing in the absence and presence of organic materials under different exposure times, such as 5 s, 30 s, 1 min, 3 min, 5 min, 10 min, and 15 min. AIV inactivation was assessed on contaminated carrier surfaces, such as stainless steel, rubber, plastic, and artificially contaminated clothes. Results: In aqueous phase, concentrations of 1×, 0.5×, 0.25×, and 0.125× inactivated NDV in the absence of organic materials within 5 s, 5 s, 5 min, and 15 min at concentrations of 1×, 0.5×, 0.25×, and 0.125×, respectively. In the presence of organic material contamination, NDV could be inactivated within 30 s for 1×, 1 min for 0.5×, and 10 min for 0.25×; however, 0.125× PPMS did not achieve inactivation within 15 min. PPMS concentrations of 1×, 0.5×, 0.25×, and 0.125× inactivated AIV within 5 s, 5 s, 5 s, and 30 s, respectively, in both the absence and presence of organic materials. PPMS at a concentration of 1× could inactivate AIV on all carriers within 30 s. PPMS at 0.5× and 0.25× concentrations could inactivate AIV within 30 s on rubber and plastic; inactivation occurred within 1 min on stainless steel. However, 0.125× PPMS and 1× QAC could not achieve inactivation within 3 min on all carriers. Finally, PPMS concentrations of 1×, 0.5×, 0.25×, and 0.125× inactivated AIV on rayon sheets within 5 s, 30 s, 5 min, and 15 min, respectively. However, the recommended NaDCC concentration achieved inactivation within 10 min, whereas QAC did not achieve inactivation within 15 min. Conclusion: PPMS can inactivate enveloped viruses such as NDV and AIV. Furthermore, PPMS is superior to NaDCC and QAC for inactivating viruses on various carrier surfaces and artificially contaminated clothes. However, the virucidal efficacy of PPMS depends on the optimal concentration, organic material conditions, and exposure/contact timing. Therefore, PPMS is a promising alternative disinfectant crucial for enhancing biosecurity and controlling viruses that contaminate animal farms, slaughterhouses, and hospitals.

H10 subtype avian influenza viruses (AIV) have been circulating in China for 40 years. H10 AIVs in China have expanded their host range from wild birds to domestic poultry and mammals, even human. Most of the H10 subtype AIVs reported in China were isolate from the southeast part. We isolated an H10N3 AIV, A/Chicken/Liaoning/SY1080/2021 (SY1080), from live poultry market (LPM) in Liaoning Province of the Northeast China. SY1080 replicated efficiently in mice lungs and nasal turbinates without prior adaptation. We systematically compared SY1080 with other H10 subtype isolates in China. Phylogenetic analysis showed that SY1080 and most of the H10 strains belonged to the Eurasian lineage. H10 AIVs in China have formed 63 genotypes. SY1080 as well as the H10N3 strains from human infections belonged to G60 genotype. H10Nx AIV acquired multiple mammalian adaptive and virulence related mutations during circulation and the recent reassortants derived internal genes from chicken H9N2 AIVs. The H10Nx subtypes AIVs posed potential threat to public health. These results suggested we should strengthen the surveillance and evaluation of H10 subtype strains.

Background and Aim: Avian influenza is a global threat to avian species, particularly in developing countries. Recombinant vaccines, including virus-like particles (VLPs), are promising strategies for preventing the spread of the disease. VLPs produced through the self-assembly of viral structural proteins without genomic material mimic native virions and are promising platforms for new vaccines. VLPs have been shown to elicit protective antibodies and are effective and safe vaccines against influenza. This study aimed to optimize the protocol for the production and characterization of H9N2 VLPs and their evaluation as a vaccine in broiler birds. Materials and Methods: Low-pathogenic influenza virus (LPAI) H9N2 was isolated and characterized through whole-genome sequencing, and a VLP-based vaccine for LPAI H9N2 was prepared using a baculovirus expression system. Codon-optimized hemagglutinin (HA), neuraminidase (NA), and M1 were successfully cloned in pFastbac1 and expressed in SF9 cells. Proteins were characterized using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy after purification. Semi-purified proteins were tested as a vaccine in broiler chickens challenged with LPAI H9N2. Results: Recombinant Bacmid DNA from positive clones was extracted and confirmed using a polymerase chain reaction. The transfection showed cytopathic effects, and the proteins were confirmed through western blotting and SDS-PAGE, which showed the sizes of HA = 62–64 KD, NA = 52 KD, and M1 = 25 KD. The shape and morphology were confirmed through transmission electron microscopy which revealed 100–150 nm size particles. As a result, the semi-purified VLPs (HA assay: 256) were tested as a vaccine for specific-pathogen free broiler birds; administered through subcutaneous and intranasal routes. The birds were challenged on the 28th day after vaccination with the H9N2 strain, and the birds showed significant cross-reactivity with the H9N2 strain. Conclusion: The semi-purified VLP-based vaccine induced a significant immune response in vivo. This vaccine formulation has the potential to control avian influenza outbreaks in Pakistan’s poultry industry.

Relevance. Influenza A virus is characterized by a segmented single-stranded RNA genome. Such organization of the virus genome determines the possibility of reassortment, which can lead to the emergence of new virus variants. The main natural reservoir of most influenza A virus subtypes are wild waterfowl. Seasonal migrations gather waterfowl from all major migration routes to nesting areas near the northern and southern polar circles. This makes intercontinental spread of influenza A viruses possible. Objective ‒ to conduct molecular genetic monitoring and study the phylogenetic relationships of influenza A virus variants circulating in Antarctica in 2023. Materials and methods. We studied 84 samples of biological material obtained from birds and marine mammals in April‒May 2023 in coastal areas of Antarctica. For 3 samples, sequencing was performed on the Miseq, Illumina platform and phylogenetic analysis of the obtained nucleotide sequences of the influenza A virus genomes was performed. Results. The circulation of avian influenza virus in the Antarctic region was confirmed. Heterogeneity of the pool of circulating variants of the influenza A virus (H3N8, H1N1) was revealed. Full-length genomes of the avian influenza virus were sequenced and posted in the GISAID database (EPI_ISL_19032103, 19174530, 19174467). Conclusion. The study of the genetic diversity of influenza A viruses circulating in the polar regions of the Earth and the identification of the conditions for the emergence of new genetic variants is a relevant task for the development of measures to prevent biological threats.

The H2N2 avian influenza viruses (AIV) have been reported in the Northeast United States of America (USA) live bird market (LBM) system since 2014. In this study, we investigated the genetic evolution and characterized molecular markers of the recent H2N2 AIVs in LBMs in the Northeast USA. Phylogenetic analyses revealed that the LBM H2N2 lineage has evolved into three distinct subgroups (groups A.1, A.2, and A.3). The group A.1 viruses and some transient reassortants evolved through several independent reassortment events between the LBM H2N2 lineage and North American wild bird-origin AIVs. Separately, a group of phylogenetically distinct novel H2N2 viruses (group B) identified in LBMs completely originated from wild birds, independent from the previous LBM H2N2 lineage that has persisted since 2014. While no molecular evidence of mammalian adaptation was found, the novel H2N2 viruses in the LBM system underscore the importance of updated risk assessments for potential human transmission. [ABSTRACT FROM AUTHOR]

Three subtypes of Avian Influenza Viruses (AIVs)–H1N1, H9N2, and H5N2–were detected in 1.4 THz using metamaterial absorptive biosensor. The proposed sensor was consisted of an array of gold cross-shaped resonator on a silica-gold–silicon wafer. The sensor operates based on the interaction between the virus-infected cells and incident electromagnetic (EM) waves under room temperature condition, resulting in resonance peaks in the absorption spectrum within the terahertz frequency range of 1–2THz. The results were simulated using finite element method (FEM), achieving an ultra-high sensitivity of 53.6 µm/RIU, which corresponds to a figure of merit as high as 28.84. The proposed sensor holds great potential in reducing economic damages in the poultry industry, ensuring public health safety and can also be utilized for the identification of other biological agents or environmental contaminants. [ABSTRACT FROM AUTHOR]

Avian influenza (AI) is an extremely contagious viral disease of domestic and wild birds that can spread rapidly among bird populations, inducing serious economic losses in the poultry industry. During the winter season 2021–2022, we isolated seventeen highly pathogenic avian influenza (HPAI) H5N8 viruses from outbreaks involving ducks in Egypt, occurring in both backyard and farm settings. The aim of this study was to pinpoint genetic key substitutions (KSs) that could heighten the risk of a human pandemic by influencing the virus's virulence, replication ability, host specificity, susceptibility to drugs, or transmissibility. To understand their evolution, origin, and potential risks for a human pandemic, whole-genome sequencing and phylogenetic analysis were conducted. Our analysis identified numerous distinctive mutations in the Egyptian H5N8 viruses, suggesting potential enhancements in virulence, resistance to antiviral drugs, and facilitation of transmission in mammals. In this study, at least five genotypes within one genome constellation of H5N8 viruses were identified, raising concerns about the potential emergence of novel viruses with altered characteristics through reassortment between different genotypes and distinct groups. These findings underscore the role of ducks in the virus's evolutionary process and emphasize the urgent need for enhanced biosecurity measures in domestic duck farms to mitigate pandemic risk. [ABSTRACT FROM AUTHOR]

Genetically diverse avian influenza viruses (AIVs) are maintained in wild aquatic birds with increasingly frequent spillover into mammals, yet these represent a small proportion of the overall detections. The isolation of AIVs in marine mammals, including seals, has been reported sporadically over the last 45 years. Prior to 2016, all reports of AIVs detected in seals were of low-pathogenicity AIVs. In spite of this, the majority of reported AIV outbreaks caused fatal respiratory diseases, with harbor seals particularly susceptible to infection. The H5 clade 2.3.4.4b highly pathogenic AIV (HPAIV) was detected in seals for the first time in 2016. Recently, many cases of mass seal die-offs have occurred because of 2.3.4.4b HPAIV and are attributed to spillover from wild bird species. The potential for seal-to-seal transmission has been considered after the mass mortality of southern elephant seals off the coast of Argentina. Close contact between seals and wild birds, the rapid evolution of H5N1 AIVs, and the possibility of efficient mammal-to-mammal transmission are increasing concerns due to the potential for the establishment of a marine mammal reservoir and public health risks associated with the pandemic potential of the virus. This manuscript details the detection of AIVs in the seal population, comparing interesting features of various subtypes with an emphasis on avian-to-mammal-to-mammal transmission. Phylogenetic characterizations of the representative seal isolates were performed to demonstrate the relationships within the different virus isolates. Furthermore, we demonstrate that the reassortment events between different LPAIVs occurred before and after the viruses reached the seal population. The reassortment of viral segments plays an important role in the evolution of influenza viruses. Taken together, these data report on the 45 year history between seals and AIVs. [ABSTRACT FROM AUTHOR]

In January 2022, six gibbons (Nomascus spp.) in the same enclosure of a zoo successively showed symptoms of runny nose and cough. We separately collected six nasal swabs from the six gibbons, detected the pathogens in the samples by high-throughput sequencing technology. The results showed that two samples were positive for AIV. The sequence assembly was similar to H9N2 avian influenza virus. The two positive samples were tested by qPCR for H1 - H16 genes and N1 - N9 genes and the results showed that Ct values of H9 and N2 in sample 5 were 31. 35 and 36. 16 respectively, Ct values of H9 and N2 in sample 6 were 27. 46 and 29. 57. The gibbons were given comprehensive treatment, such as expectoration and cough relief, anti-inflammatory and secondary infection control, and disinfection of the feeding environment was strengthened. The sick gibbons recovered quickly. [ABSTRACT FROM AUTHOR]

Copyright of Avian Diseases is the property of American Association of Avian Pathologists, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

ABSTRACT Following two human infections with the H10N3 avian influenza virus (AIV) in 2021 and 2022, a third case was discovered in Yunnan, China, in 2024, raising concerns about the potential for future pandemics. Recent studies have indicated that novel H10N3 viruses are highly pathogenic in mice and can be transmitted between guinea pigs via respiratory droplets without prior adaptation. However, the biological characteristics of novel H10N3 in poultry have not been fully elucidated. Our findings revealed that H10 subtype AIVs are predominantly prevalent in waterfowl. Notably, H10N8 and H10N3 viruses that have infected humans were primarily isolated from chickens. For the first time, double basic hemagglutinin cleavage sites (motif PEIKQGR↓GL) were identified in novel H10N3 AIVs, which exhibit enhanced replication in chickens, and can be transmitted between chickens through direct contact and respiratory droplets. Animal experimental studies demonstrated that ducks are also susceptible to H10N3 viruses and that the virus is transmissible through direct contact, suggesting a greater risk of transmission and recombination. Serological studies conducted among poultry workers suggest that while the human population was largely naïve to H10N3 infection, sporadic and undetected human infections did occur, indicating a potential increasing trend. These data further emphasize the growing threat to public health posed by zoonotic H10N3 subtype AIVs.IMPORTANCEExposure to poultry in live poultry markets (LPMs) is strongly associated with human infection with avian influenza viruses (AIVs), with chickens being the most common species found in these markets in China. The prevalence of AIVs in chickens, therefore, increases the risk of human infection. Notably, the main host of the novel H10N3 virus has shifted from waterfowl to chickens, and the virus can be transmitted between chickens via respiratory droplets, posing a potential risk of a pandemic within poultry populations. The novel H10N3 virus also remains sensitive to ducks and can be transmitted through direct contact, which means a greater risk of transmission and recombination. Significantly, the human population remains largely naïve to H10N3 infection, but sporadic seropositivity among poultry workers indicates previous exposure to H10 subtype AIVs. Therefore, a comprehensive surveillance of the novel H10N3 viruses in poultry is imperative. Effective control of the virus within poultry populations could significantly reduce the risk of emerging human infections.

Rapid and accurate detection of H5Nx avian influenza viruses is critical for effective surveillance and control measures. Currently, RT-qPCR with spin column RNA extraction is the gold standard for HPAIV surveillance, but its long reaction time and need for specialized equipment limit its effectiveness for rapid response. In this study, we introduce a centrifuge-free, rapid detection method for on-site detection of H5Nx viruses that combines magnetic bead-based ribonucleoprotein (RNP) purification and concentration with a CRISPR-Cas12a system that is independent of the protospacer adjacent motif (PAM) sequence. Our approach employs anti-NP monoclonal antibodies for the targeted isolation of RNA bound to RNPs, facilitating a quick and specific RNA extraction process that negates the need for centrifugation. Additionally, by denaturing the RT-RPA amplicon using 60% DMSO, we activate the trans-ssDNA cleavage activity of the Cas12a protein without the need for a specific PAM (5’-TTTV-3′) sequence. This strategy increases flexibility in CRISPR RNA design, providing a significant advantage when targeting genes with high variability. We validated the efficacy of our magnetic RNP purification and concentration method in combined with an RT-RPA/PAM-independent Cas12a assay for detecting the H5 gene. The assay achieved a sensitivity threshold of 101 EID50 with fluorescent detection and 102 EID50 using lateral flow strips. It also exhibited high specificity, yielding positive results solely for H5Nx viruses among various influenza A virus subtypes. Furthermore, in clinical samples, the assay demonstrated 80% sensitivity and 100% specificity. These results highlight the advantages of using NP-specific antibodies for RNP purification and CRISPR-Cas12a with viral gene-specific crRNA to achieve exceptional diagnostic specificity.

The H9N2 subtype of avian influenza virus (AIV) is widely distributed among poultry and wild birds and is also a threat to humans. During AIV active surveillance in Liaoning province from 2015 to 2016, we identified ten H9N2 strains exhibiting different lethality to chick embryos. Two representative strains, A/chicken/China/LN07/2016 (CKLN/07) and A/chicken/China/LN17/2016 (CKLN/17), with similar genomic background but different chick embryo lethality, were chosen to evaluate the molecular basis for this difference. A series of reassortants between CKLN/07 and CKLN/17 were generated and their chick embryo lethality was assessed. We found that the isoleucine (I) residue at position 137 (H3 numbering) in the hemagglutinin (HA) was responsible for the chick embryo lethality of the H9N2 virus. Further studies revealed that the threonine (T) to I mutation at HA position 137 enhanced viral replication in vitro and in vivo. Moreover, the HA-T137I substitution in H9N2 avian influenza virus increased the guinea pig transmission efficiency. We also found that the HA-T137I substitution was critical for α2,6-linked sialic acid binding preference and HA activation and stability of H9N2 virus. Our findings demonstrated that HA-137I is a key molecular marker for mammalian adaptation of H9N2 AIV.

H4Nx avian influenza viruses (AIVs) have been isolated from wild birds and poultry and can also cross the species barrier to infect mammals (pigs and muskrats). The widespread presence of these viruses in wild birds and poultry and their ability to be transmitted interspecies make them an undeniable hazard to the poultry farming industry. In the present study, we collected fecal and swab samples from wild birds and poultry in Guangdong Province from January 2019 to March 2024, and various subtypes of AIVs were isolated, including 19 strains of H4 subtype AIVs. Further analysis was conducted on the internal genes of the 19 strains. These strains clustered together with high homology to highly pathogenic avian influenza virus (HPAIV), suggesting that H4Nx AIV may be reassorted from HPAIV. Two H4N8 strains are phylogenetically related to the porcine H4N8 AIV. Molecular characterization revealed that all viruses in this study were less pathogenic but had potential mammalian-adapted mutations. The transmission dynamics of H4Nx AIVs revealed that Europe and Asia, especially the Netherlands and Bangladesh, may be the centers of transmission. This may be linked to the migration of wild birds. The high migration rates from Russia to the Netherlands and from Russia to Bangladesh may also play a role. Therefore, continuous and systematic monitoring of wild birds to clarify the spatial and temporal distribution and prevalence of influenza viruses in wild birds is significant for early warning of avian influenza outbreaks in poultry and for risk assessment for public health and safety.

H5N1 avian influenza viruses bearing the clade 2.3.2.1 hemagglutinin (HA) gene have been widely detected in birds and poultry in several countries. During our routine surveillance, we isolated 28 H5N1 viruses between January 2017 and October 2020. To investigate the genetic relationship of the globally circulating H5N1 viruses and the biological properties of those detected in China, we performed a detailed phylogenic analysis of 274 representative H5N1 strains and analyzed the antigenic properties, receptor-binding preference, and virulence in mice of the H5N1 viruses isolated in China. The phylogenic analysis indicated that the HA genes of the 274 viruses belonged to six subclades, namely clades 2.3.2.1a to 2.3.2.1f; these viruses acquired gene mutations and underwent complicated reassortment to form 58 genotypes, with G43 being the dominant genotype detected in eight Asian and African countries. The 28 H5N1 viruses detected in this study carried the HA of clade 2.3.2.1c (two strains), 2.3.2.1d (three strains), or 2.3.2.1f (23 strains), and formed eight genotypes. These viruses were antigenically well-matched with the H5-Re12 vaccine strain used in China. Animal studies showed that the pathogenicity of the H5N1 viruses ranged from non-lethal to highly lethal in mice. Moreover, the viruses exclusively bound to avian-type receptors and have not acquired the ability to bind to human-type receptors. Our study reveals the overall picture of the evolution of clade 2.3.2.1 H5N1 viruses and provides insights into the control of these viruses.

ABSTRACTHuman infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid “aa” in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Several genotypes of the highly pathogenic avian influenza (HPAI) virus H5N8 subtype within clade 2.3.4.4b continue to circulate in different species of domestic birds across Egypt. It is believed that quail contribute to virus replication and adaptation to other gallinaceous poultry species and humans. This study provides genetic characterization of the full genome of HPAI H5N8 isolated from quail in Egypt. The virus was isolated from a commercial quail farm associated with respiratory signs. To characterize the genetic features of the detected virus, gene sequencing via Sanger technology and phylogenetic analysis were performed. The results revealed high nucleotide identity with the HPAI H5N8 virus from Egypt, which has multiple basic amino acid motifs PLREKRRKR/GLF at the hemagglutinin (HA) cleavage site. Phylogenetic analysis of the eight gene segments revealed that the quail isolate is grouped with HPAI H5N8 viruses of clade 2.3.4.4b and closely related to the most recent circulating H5N8 viruses in Egypt. Whole-genome characterization revealed amino acid preferences for avian receptors with few mutations, indicating their affinity for human-like receptors and increased virulence in mammals, such as S123P, S133A, T156A and A263T in the HA gene. In addition, the sequencing results revealed a lack of markers associated with influenza antiviral resistance in the neuraminidase and matrix-2 coding proteins. The results of the present study support the spread of HPAIV H5N8 to species other than chickens in Egypt. Therefore, continuous surveillance of AIV in different bird species in Egypt followed by full genomic characterization is needed for better virus control and prevention.

The H9N2 subtype of avian influenza virus (AIV) is widely prevalent in poultry and wild birds globally, and has become the predominant subtype circulating in poultry in China. The H9N2 AIV can directly or indirectly (by serving as a “donor virus”) infect humans, posing a significant threat to public health. Currently, there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province, central China. In this study, we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province, and genetic analysis revealed that these viruses belonged to 7 different genotypes. Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations, and exhibited diverse replication abilities within these birds. These viruses could replicate efficiently in the lungs of mice, with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys. At the cellular level, the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays, revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level. The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters. Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China. It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Background: Poultry industries play an important role in the human food supply. Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), and Avian influenza virus (AIV) can cause epizootic outbreaks in poultry industries and lead to extensive economic losses. Furthermore, these viruses can also infect wild birds, and avian influenza virus is a serious threat to humans. Here, a multiepitope vaccine has been designed to induce an immune response against IBV, NDV, and AIV using extensive bioinformatics tools. Methods: To do so, the antigenic proteins, including the hemagglutinin of H5 and H7 subtypes of AIV, nucleocapsid and spike proteins of IBV, and fusion and hemagglutinin-neuraminidase proteins of NDV, have been studied to find immunodominant epitopes with MHC-I/MHC-II binding potential. Four antigenic and non-allergenic epitopes from each antigenic protein were connected to avian beta-defensin 1 as an adjuvant to construct the multiepitope vaccine. To investigate the potential of vaccine-induced activation of toll-like receptors (TLR-2, 5), the tertiary structure of the vaccine was modeled and docked to TLR-2/5 proteins. Results: Evaluation of the physicochemical properties of the vaccine construct has demonstrated the stability and solubility of the vaccine upon overexpression. The vaccine construct demonstrated antigenicity and was specified as a non-allergenic protein. The vaccine can induce significant cellular and humoral immune responses, and TLR proteins can recognize the vaccine in its three-dimensional form. Conclusion: Overall, the multiepitope vaccine designed in the present study against IBV, NDV, and AIV shows significant immunological potential that should be further investigated in wet laboratory experiments.

The year 2023 saw a challenging epizootiological and epidemiological situation regarding highly pathogenic avian influenza. The virus affected 150 bird species and dozens of mammal species. More than 650 outbreaks were reported in poultry across 29 countries, resulting in the death or destruction of approximately 19 million specimens. There was a high incidence of the influenza among wild birds (approximately 3,000 outbreaks in 65 countries) and mammals (more than 16,000 cases). The majority of outbreaks in wild birds, poultry and mammals were caused by influenza A(H5N1) clade 2.3.4.4b viruses. Many countries in Europe, Asia, Africa, North and South America experienced the outbreaks throughout the year. For the first time, a polar bear death from A(H5N1) virus was documented. Moreover, molecular markers of virus adaptation to mammals were found in PB2 proteins of 50 % of influenza A(H5N1) viruses that caused the death of animals. During the year 2023, human infections with highly pathogenic avian influenza A(H5N1) viruses were reported in Cambodia, Chile, China, and the UK. In addition, human infections with A(H3N8), A(H5N6), A(H9N2) and A(H10N5) viruses were reported in China. In Russia in 2023, outbreaks among wild birds and poultry were registered in 25 regions, as well as an outbreak among fur seals in the Sakhalin Region. The stated outbreaks were caused by highly virulent influenza A(H5N1) clade 2.3.4.4b. Hemagglutinin sequences of all Russian viruses analyzed in this study in 2023 were genetically close to the WHO candidate vaccine strains A/Astrakhan/3212/2020 (H5N8), A/chicken/ Ghana/AVL-763_21VIR7050-39/2021 (H5N1) and A/American Wigeon/South Carolina/22-000345-001/2021 (H5N1). All studied A(H5N1) viruses were antigenically similar to the A/Astrakhan/3212/2020 vaccine strain.

We detected antibodies to H5 and N1 subtype influenza A viruses in 4/194 (2%) dogs from Washington, USA, that hunted or engaged in hunt tests and training with wild birds. Historical data provided by dog owners showed seropositive dogs had high levels of exposure to waterfowl.

We isolated novel reassortant avian influenza A(H5N6) viruses containing genes from clade 2.3.4.4b H5N1 virus and low pathogenicity avian influenza viruses in carcasses of whooper swans and bean geese in South Korea during December 2023. Neuraminidase gene was from a clade 2.3.4.4b H5N6 virus infecting poultry and humans in China.

Based on the pathogenicity in chickens, most H1–H16 avian influenza viruses (AIV) cause mild diseases, whereas some of the H5 and H7 AI viruses cause severe, systemic disease. The number of basic amino acids in the haemagglutinin (HA) cleavage site of AIV plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (intravenous pathogenicity index [IVPI] and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in the absence of an in vivo study determination. This approach not only expedites the declaration process of highly pathogenic AIV (HPAIV) but also reduces the need for experimental in vivo testing of H5 and H7 viruses. [ABSTRACT FROM AUTHOR]

Accepting the fact that there is a huge number of virus particles in food that lead to several infectious diseases, eliminating of the foodborne virus from food is tangible. In 2020, the appearance of new SARS-CoV-2 variants had remarked the importance of food safety in our lives. Detection virus is a dynamic domain. Recently, many papers have tried to detect several foodborne viruses by using conventional sensing platforms including ELISA (enzyme-linked immunosorbent assay), PCR (polymerase chain reaction-based methods) and NASBA (nucleic acid sequence-based amplification). However, small sizes, low infective doses and discrete distribution of the foodborne virus have converted these microorganisms into the most challengeable pathogen in the food samples matrix. Foodborne virus detection exploiting aptamer-based biosensors has attracted considerable attention toward the numerous benefits of sourcing from aptamers in which a variety of viruses could be detected by conjugation of aptamer-virus. The development of multiple sensing methodologies and platforms in terms of aptasensor application in real food and environment samples has demonstrated promising results. In this review, we present the latest developments in myriad types of aptasensors (including electrochemical, optical and piezoelectric aptasensor) for the quantification of foodborne viruses. Working strategies, benefits and disadvantages of these platforms are argued. [ABSTRACT FROM AUTHOR]

The evolution of the H5 highly pathogenic avian influenza (HPAI) viruses has led to the emergence of distinct groups with genetically similar clusters of hemagglutinin (HA) sequences. In this study, a consensus H5 HA sequence was cloned into the baculovirus expression system. The HA protein was expressed in baculovirus-infected insect cells and utilized as the antigen for the production of an oil emulsion-based H5 avian influenza vaccine (rBacH5Con5Mut). Twenty-one-day-old SPF chickens were immunized with this vaccine and then challenged at 21 days post-vaccination with clade 2.3.2.1, clade 2.3.4.4, and clade 7.2 of H5 HPAI viruses. The sera of vaccinated chickens exhibited high hemagglutination inhibition (HI) titers against the rBacH5 vaccine antigen, while lower HI titers were observed against the different challenge virus H5 hemagglutinins. Furthermore, the rBacH5Con5Mut vaccine provided 100% protection from mortality and clinical signs. Virus isolation results showed that oropharyngeal and cloacal shedding was prevented in 100% of the vaccinated chickens when challenged with clade 2.3.2.1 and clade 2.3.4.4 H5 viruses. When the rBacH5Con5Mut vaccine candidate was administrated at one day of age, 100% protection was demonstrated against the challenge of clade 2.3.4.4 virus at three weeks of age, indicating the potential of this vaccine for hatchery vaccination. Overall, A single immunization of rBacH5Con5Mut vaccine candidate with a consensus HA antigen can protect chickens against different clades of H5 HPAI viruses throughout the rearing period of broiler chickens without a boost, thus fulfilling the criteria for an efficacious broad-spectrum H5 avian influenza vaccine. [ABSTRACT FROM AUTHOR]

Unidentified diseases are becoming more prevalent among humans due to various climatic factors, and some of these diseases originate in animals before spreading to humans. One virus that has been of particular concern is the avian influenza virus, which primarily infects bird and can subsequently transmit to humans. This article presents a mathematical model describing the spatio-temporal reaction-diffusion process involved in the transmission of avian flu in human population. The paper begins by studying the proposed model's well-posedness and the calculated basic reproduction number, which provides valuable insights into the dynamics of virus transmission. The paper also provides stability analysis for the disease-free steady state of the model. All theoretical studies are validated using computational results. [ABSTRACT FROM AUTHOR]

Background In recent years, Vietnam has suffered multiple epizootics of influenza in poultry. Methods From 10 January 2019 to 26 April 2021, we employed a One Health influenza surveillance approach at live bird markets (LBMs) and swine farms in Northern Vietnam. When the COVID-19 pandemic permitted, each month, field teams collected oral secretion samples from poultry and pigs, animal facility bioaerosol and fecal samples, and animal worker nasal washes at 4 LBMs and 5 swine farms across 5 sites. Initially samples were screened with molecular assays followed by culture in embryonated eggs (poultry swabs) or Madin-Darby canine kidney cells (human or swine swabs). Results Many of the 3493 samples collected had either molecular or culture evidence for influenza A virus, including 314 (37.5%) of the 837 poultry oropharyngeal swabs, 144 (25.1%) of the 574 bioaerosol samples, 438 (34.9%) of the 1257 poultry fecal swab samples, and 16 (1.9%) of the 828 human nasal washes. Culturing poultry samples yielded 454 influenza A isolates, 83 of which were H5, and 70 (84.3%) of these were highly pathogenic. Additionally, a positive human sample had a H9N2 avian-like PB1 gene. In contrast, the prevalence of influenza A in the swine farms was much lower with only 6 (0.4%) of the 1700 total swine farm samples studied, having molecular evidence for influenza A virus. Conclusions This study suggests that Vietnam's LBMs continue to harbor high prevalences of avian influenza A viruses, including many highly pathogenic H5N6 strains, which will continue to threaten poultry and humans. [ABSTRACT FROM AUTHOR]