Avian Influenza Virus Strains Research Articles

Avian influenza mRNA vaccine encoding hemagglutinin provides complete protection against divergent H5N1 viruses in specific-pathogen-free chickens

BackgroundThe rapid mutation of avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Herein, we have successfully developed an mRNA-LNPs candidate vaccine for H5 subtype highly pathogenic avian influenza and evaluated its immunogenicity and protective efficacy.ResultsIn experiments on BALB/c mice, the vaccine candidate elicited strong humoral and a certain cellular immune responses and protected mice from the heterologous AIV challenge. Antibody and splenocyte passive transfer assays in mice suggested that antibodies played a crucial role in providing protection. Experiments involving SPF chickens have revealed that two doses of the 5 µg vaccine candidate in this study provided 100% complete protection against homologous strains, but only 50% complete protection against heterologous strains. Even immunization with two doses of the 15 µg vaccine candidate resulted in 90% complete protection against heterologous strains. To enhance the immune efficacy of the candidate vaccine, we designed 6 sequences with different secondary structures and screened out the candidate sequence with the highest expression (SY2-HA mRNA). Experiments on SPF chickens showed that two doses of 5 µg SY2-HA mRNA-LNP vaccine provided 100% complete protection against homologous and heterologous H5N1 AIV strains. Immunization tests with the SY2-HA mRNA-LNP vaccine were repeated in the SPF chicken model, inducing antibody production levels that are consistent with previous tests and providing 100% complete protection against both homologous and heterologous strains of the virus, indicating that the vaccine has a stable immune efficacy.ConclusionsThe vaccine developed in this study provides complete protection against divergent H5N1 AIV strains in chickens, offering a promising approach for the future development of mRNA vaccines against multivalent avian influenza subtypes.Graphical

Geographical distribution and evolutionary dynamics of H4Nx avian influenza viruses.

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.

OPTIMIZATION OF THE EXTRACTION PROCESS OF BIOLOGICALLY ACTIVE COMPLEXES FROM PSORALEA DRUPACEA BGE ROOTS

In recent years, due to the emergence of previously unknown types of viral diseases (avian influenza, swine flu type A/H1N1), the search for new drugs against bacteria and viruses has become a very pressing problem. Synthetic drugs, which, in addition to the obvious antiviral and antiviricidal action, have a number of side effects on the nervous, respiratory, digestive, hepatobiliary systems, and also cause allergic reactions as a result predominate among modern drugs in this field. In terms of side effects of herbal medicines, the advantage is to reduce the effects, since the biosynthesis of plant-based drugs does not contradict the biosynthesis of humans and animals. In the course of solving these current problems, it has been established that the dry extract obtained from the roots of Psoralea drupacea Bge. is active against strains of avian influenza viruses. The article presents the results of research related to the optimization of the extraction process of this substance. The influence of various factors of the extraction process: extractant nature, raw material grinding degree, extraction duration, extraction method (sedimentation (maceration), percolation, extraction with a boiling solvent in a Soxhlet apparatus) on the total yield of extractive substances and the amount of furocoumarins was studied. The results of the research demonstrated that the most effective extractant is ethanol, and the optimal degree of raw material grinding is 1.25 mm. The most optimal extraction method is raw material extraction with ethanol by Soxhlet apparatus. In this case, after 3 days, the dry extract yield was high (5.80%) and the maximum amount of furocoumarins was also high (72.34%). Nevertheless, maceration with stirring and heating can give good results, and the advantage of the process is its simplicity and the ability to increase the raw material upload.

H5 subtype avian influenza virus induces Golgi apparatus stress response via TFE3 pathway to promote virus replication.

During infection, avian influenza virus (AIV) triggers endoplasmic reticulum (ER) stress, a well-established phenomenon in previous research. The Golgi apparatus, situated downstream of the ER and crucial for protein trafficking, may be impacted by AIV infection. However, it remains unclear whether this induces Golgi apparatus stress (GAS) and its implications for AIV replication. We investigated the morphological changes in the Golgi apparatus and identified GAS response pathways following infection with the H5 subtype AIV strain A/Mallard/Huadong/S/2005. The results showed that AIV infection induced significant swelling and fragmentation of the Golgi apparatus in A549 cells, indicating the presence of GAS. Among the analyzed GAS response pathways, TFE3 was significantly activated during AIV infection, while HSP47 was activated early in the infection process, and CREB3-ARF4 remained inactive. The blockade of the TFE3 pathway effectively inhibited AIV replication in A549 cells and attenuated AIV virulence in mice. Additionally, activation of the TFE3 pathway promoted endosome acidification and upregulated transcription levels of glycosylation enzymes, facilitating AIV replication. These findings highlight the crucial role of the TFE3 pathway in mediating GAS response during AIV infection, shedding light on its significance in viral replication.

Serological response to avian influenza viruses among occupationally exposed population in Taiwan, a longitudinal cohort study, 2021-2023.

The Taiwan Centers for Disease Control (TCDC) initiated longitudinal sero-epidemiological surveillance of avian influenza viruses (AIVs) targeting poultry workers exposed on affected farms since 2021. Convalescent-phase serum samples were collected from these workers 21-28 days post-outbreak identification. TCDC recontacted participants in subsequent years for repeat serological sampling. Hemagglutination inhibition (HI) assays were used to test against representative strains of AIV A/H5 2.3.4.4c and A/H5 2.3.4.4b. By the end of 2023, a 2-year cohort of 59 participants and a 3-year cohort of 61 participants were established. None of them had laboratory-confirmed infection. HI titers against AIV A/H5 2.3.4.4c remained low from 2021 to 2023. For AIV A/H5 2.3.4.4b, HI titer remained low from 2021 to 2022 but increased in more than half of the participants (60%, 72/120) in 2023. This change parallels the increasing AIV A/H5 2.3.4.4b burden indicated by increased outbreaks and warrants continuous monitoring.

Molecular diagnostics using the QIAstat-Dx syndromic device for covering avian influenza pandemic preparedness

IntroductionA key factor in influenza pandemic preparedness is the ability to detect zoonotic influenza virus strains as they emerge in humans through spillover events, ideally before human-to-human transmission occurs. DesignIn this study, the utility of the QIAstat-Dx syndromic device for influenza surveillance was evaluated. Bioinformatic analysis was performed on all WHO-recommended influenza Candidate Vaccine Viruses (CVVs), including the common strains recommended for the 2023–2024 influenza vaccine composition in the northern hemisphere, and 16 different H5 highly pathogenic avian influenza virus (HPAIV) and two H9N2 low pathogenic avian influenza virus (LPAIV) strains. For laboratory validation, engineered gene fragments and real HPAIV and LPAIV samples were tested using the QIAstat-Dx Respiratory SARS-CoV-2 Panel. ResultsDuring the bioinformatic screening, common influenza strains were positive including influenza A subtypes, and all H5 HPAIV and LPAIV H9N2 were detected as Influenza A positive without subtype discrimination. In all cases, laboratory validation confirmed all bioinformatic results. ConclusionQIAstat-Dx can detect all tested potentially zoonotic influenza A virus strains, and discriminate them from human sesonal influenza A viruses, ensuring a correct diagnosis. Any tool available for surveillance and pandemic preparedness is essential for a rapid response to reduce healthcare costs and severity of future influenza pandemics.

The highly pathogenic avian influenza H5N8 sublineage 2.3.4.4b virus: A critical analysis of genetic steadiness based on full HA gene sequencing in Egypt

The highly pathogenic avian influenza virus (HPAIV) continues to be a significant threat to the poultry industry despite the implementation of vaccination programs. The main goal of this study was to explore the incidence of avian influenza viruses (AIVs) and the stability of the hemagglutinating (HA) gene sequence. For this drive, oropharyngeal swabs (n=425) were collected from various wild and domesticated bird species from 13 provinces in Egypt from 2019 to 2021. Viral isolation in specific pathogen-free chicken embryos revealed positive HA activity in 9.88% (n=32) of the wild and 36.37% (n=64) of domesticated species. Only eight H5N8 avian influenza viruses were delivered (4/each category) in mixed infection with Newcastle disease virus (NDV). Clinically, domesticated examined birds exhibited clinical findings suggesting avian influenza, but AIVs were delivered from both apparently healthy and sick wild birds. A/chicken-cobb/Egypt/55/2019(H5N8) sequence generated in this study has been submitted to GenBank under the accession number ON724339, and its in vivo pathogenicity was evaluated in four-week-old chickens, which revealed 90% mortality and typical clinical findings of HPAIV. Based on the full HA gene sequence, the strain has a Furin-sensitive cleavage site in the HA protein, which is an HPAIV. It has been clustered in clade 2.3.4.4b within AIVs from 2016 to 2023 and coexistence of genetically stable viruses 2016-2019 with 99.8-100% similarity. The existing strain is fully identical to three strains: A/green-winged teal/Egypt/877/2016(H5N8), A/northern shoveler/Egypt/MB-D-816OP/2016(H5N8), and A/duck/Egypt/N13736E/2017 (H5N8), with the only point of mutation at position 284 (G284E). In our analysis, the majority of H5NX AIV strains detected in Egypt since 2016 possessed 156T. As a result, there is evidence suggesting that H5N8 has been introduced sequentially through the migration of European wild birds along the Black Sea–Mediterranean and East Asia–West Africa flyways. This raises the possibility of future adaptation to mammals due to spillover from avian-origin strains, especially in native aquatic birds such as geese and ducks.

Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning1

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

Identification of B-cell epitopes located on the surface in the PB2 protein of the H9N2 subtype avian influenza virus

ABSTRACT Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021–2022

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015–2022 worldwide. H5N1 showed a higher rate of evolution in 2021–2022 and more sites under positive selection pressure in 2015–2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.

Avian Influenza Virus and Avian Paramyxoviruses in Wild Waterfowl of the Western Coast of the Caspian Sea (2017-2020).

The flyways of many different wild waterfowl pass through the Caspian Sea region. The western coast of the middle Caspian Sea is an area with many wetlands, where wintering grounds with large concentrations of birds are located. It is known that wild waterfowl are a natural reservoir of the influenza A virus. In the mid-2000s, in the north of this region, the mass deaths of swans, gulls, and pelicans from high pathogenicity avian influenza virus (HPAIV) were noted. At present, there is still little known about the presence of avian influenza virus (AIVs) and different avian paramyxoviruses (APMVs) in the region's waterfowl bird populations. Here, we report the results of monitoring these viruses in the wild waterfowl of the western coast of the middle Caspian Sea from 2017 to 2020. Samples from 1438 individuals of 26 bird species of 7 orders were collected, from which 21 strains of AIV were isolated, amounting to a 1.46% isolation rate of the total number of samples analyzed (none of these birds exhibited external signs of disease). The following subtypes were determined and whole-genome nucleotide sequences of the isolated strains were obtained: H1N1 (n = 2), H3N8 (n = 8), H4N6 (n = 2), H7N3 (n = 2), H8N4 (n = 1), H10N5 (n = 1), and H12N5 (n = 1). No high pathogenicity influenza virus H5 subtype was detected. Phylogenetic analysis of AIV genomes did not reveal any specific pattern for viruses in the Caspian Sea region, showing that all segments belong to the Eurasian clades of classic avian-like influenza viruses. We also did not find the amino acid substitutions in the polymerase complex (PA, PB1, and PB2) that are critical for the increase in virulence or adaptation to mammals. In total, 23 hemagglutinating viruses not related to influenza A virus were also isolated, of which 15 belonged to avian paramyxoviruses. We were able to sequence 12 avian paramyxoviruses of three species, as follows: Newcastle disease virus (n = 4); Avian paramyxovirus 4 (n = 5); and Avian paramyxovirus 6 (n = 3). In the Russian Federation, the Newcastle disease virus of the VII.1.1 sub-genotype was first isolated from a wild bird (common pheasant) in the Caspian Sea region. The five avian paramyxovirus 4 isolates obtained belonged to the common clade in Genotype I, whereas phylogenetic analysis of three isolates of Avian paramyxovirus 6 showed that two isolates, isolated in 2017, belonged to Genotype I and that an isolate identified in 2020 belonged to Genotype II. The continued regular monitoring of AIVs and APMVs, the obtaining of data on the biological properties of isolated strains, and the accumulation of information on virus host species will allow for the adequate planning of epidemiological measures, suggest the most likely routes of spread of the virus, and assist in the prediction of the introduction of the viruses in the western coastal region of the middle Caspian Sea.

A two-strain avian–human influenza model with environmental transmission: Stability analysis and optimal control strategies

On the basis of the WHO legitimated fear that there will be an avian influenza virus strain capable of mutating once it reaches the human population and sustains human-to-human transmissions, we formulate an hypothetical mathematical model which accounts for the environmental transmission and mutation of an avian influenza virus having the ability to spill over into the human population and become a highly pathogenic strain. We compute the basic reproduction number of the model and use it to study the existence and stability of equilibrium points. We derive conditions for the global asymptotic stability of any of the three equilibrium. The model is extended to incorporate six relevant time-dependent controls, and use the Pontryagin’s maximum principle to derive the necessary conditions for optimal disease control. Finally, the optimal control problem is solved numerically to show the effect of each control parameter and their combination. The incremental cost-effectiveness ratios are calculated to investigate the cost-effectiveness of all possible combinations of the control strategies. This study suggests that quarantine infected humans might be the most cost-effective strategy to control avian influenza transmissions with the virus mutation.

Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin.

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health.

H5N1 high pathogenicity avian influenza virus in migratory birds exhibiting low pathogenicity in mallards increases its risk of transmission and spread in poultry

In 2020, an H5N1 avian influenza virus of clade 2.3.4.4b was detected in Europe for the first time and was spread throughout the world by wild migratory birds, resulting in the culling of an unprecedented number of wild birds and poultry due to the epidemic. In February 2023, we isolated and identified a strain of H5N1 high pathogenicity avian influenza virus from a swab sample from a grey crane in Ningxia, China. Phylogenetic analysis of the Hemagglutinin (HA) gene showed that the virus belonged to clade 2.3.4.4b, and several gene segments were closely related to H5N1 viruses infecting humans in China. Analysis of key amino acid sites revealed that the virus contained multiple amino acid substitutions that facilitate enhanced viral replication and mammalian pathogenicity. The results of animal challenge experiments showed that the virus is highly pathogenic to chickens, moderately pathogenic to BALB/c mice, and highly infectious but not lethal to mallards. Moreover, the virus exhibited minor antigenic drift compared with the H5-Re14 vaccine strain. To this end, we need to pay more attention to the monitoring of wild birds to prevent further spread of viruses to poultry and mammals, including humans.

An amplicon-based nanopore sequencing workflow for rapid tracking of avian influenza outbreaks, France, 2020-2022.

During the recent avian influenza epizootics that occurred in France in 2020/21 and 2021/22, the virus was so contagiousness that it was impossible to control its spread between farms. The preventive slaughter of millions of birds consequently was the only solution available. In an effort to better understand the spread of avian influenza viruses (AIVs) in a rapid and innovative manner, we established an amplicon-based MinION sequencing workflow for the rapid genetic typing of circulating AIV strains. An amplicon-based MinION sequencing workflow based on a set of PCR primers targeting primarily the hemagglutinin gene but also the entire influenza virus genome was developed. Thirty field samples from H5 HPAIV outbreaks in France, including environmental samples, were sequenced using the MinION MK1C. A real-time alignment of the sequences with MinKNOW software allowed the sequencing run to be stopped as soon as enough data were generated. The consensus sequences were then generated and a phylogenetic analysis was conducted to establish links between the outbreaks. The whole sequence of the hemagglutinin gene was obtained for the 30 clinical samples of H5Nx HPAIV belonging to clade 2.3.4.4b. The consensus sequences comparison and the phylogenetic analysis demonstrated links between some outbreaks. While several studies have shown the advantages of MinION for avian influenza virus sequencing, this workflow has been applied exclusively to clinical field samples, without any amplification step on cell cultures or embryonated eggs. As this type of testing pipeline requires only a short amount of time to link outbreaks or demonstrate a new introduction, it could be applied to the real-time management of viral epizootics.

GENETIC ANALYSIS OF NUCLEOPROTEIN OF A/H3N8 INFLUENZA VIRUS OF ASIAN AND EUROPEAN ORIGIN ISOLATED IN 2018

This paper presents the results of a genetic analysis of the nucleoprotein gene (NP) of two strains of avian influenza virus isolated in the territory of the Republic of Kazakhstan in 2018 near small lakes (Alua and Zaimishche) of the North Kazakhstan region. These strains showed the presence of 54 in the nucleo-tide and 4 substitutions in the amino acid sequence, thereby significantly distancing themselves from each other. In phylogenetic analysis, the strain A/northern shoveler/North-Kazakhstan/20/2018(H3N8) showed the greatest relationship with strains from Europe, the homology between strains showed 96-97%. The second strain A/garganey/North Kazakhstan/45/2018(H3N8) showed genetic similarity with strains from Asia, the NP gene identity was 99%.

The epizootic situation of avian influenza in the Kaluga region

The article discusses the problems associated with increased virulence of strains due to the high representativeness of the sample of susceptible organisms, the spread of avian influenza and its natural reservoirs. Currently, avian influenza virus strains are divided into HPAI (highly pathogenic) and LPAI (low pathogenic). Registered outbreaks among wild and domestic birds and cases of human infection with influenza viruses of subtypes H5N8, H7N9 and H9N2 indicate an active mutation of the virus and the appearance of “aggressive” strains that can infect not only poultry, but also humans. This article analyzes the current epizootic situation of avian influenza in the Kaluga region for 2022-2023.

Prevalence of HPAI in passerines, woodpeckers and doves presented to the Center for Wild Bird Rehabilitation during the 2022 HPAI H5N1 epizootic

The Highly Pathogenic Avian Influenza (HPAI) H5N1 clade 2.3.4.4b epizootic affecting North America beginning in late 2021 has affected wild and domestic birds to an unprecedented scale. Mortalities have been documented mainly in raptors, aquatic birds (shorebirds, waterfowl), gulls and other scavenging birds, with rare reports of other species being affected. Passerines, woodpeckers, and doves are not thought to contribute significantly to the transmission of avian influenza, and thus are not regularly tested. Despite this, mortalities and even the possibility of asymptomatic carrier states in songbirds have been documented with HPAI H5N1 strains. This study focused on sampling and testing of species not typically included in HPAI surveillance, to help assess prevalence, symptoms of infection if detected and the possibility of asymptomatic carriers. Throughout two periods in 2022, 164 individuals of 38 species were sampled from Vermont and New Hampshire. None of these birds tested positive for any strain of avian influenza virus. While the scale of this study is small, it highlights the need for testing species that are not a part of regular HPAI surveillance, as well as the role rehabilitation centers may play in that goal.

Utility of Feathers for Avian Influenza Virus Detection in Commercial Poultry.

The present study evaluated the potential utility of feather samples for the convenient and accurate detection of avian influenza virus (AIV) in commercial poultry. Feather samples were obtained from AIV-negative commercial layer facilities in Iowa, USA. The feathers were spiked with various concentrations (106 to 100) of a low pathogenic strain of H5N2 AIV using a nebulizing device and were evaluated for the detection of viral RNA using a real-time RT-PCR assay immediately or after incubation at -20, 4, 22, or 37 °C for 24, 48, or 72 h. Likewise, cell culture medium samples with and without the virus were prepared and used for comparison. In the spiked feathers, the PCR reliably (i.e., 100% probability of detection) detected AIV RNA in eluates from samples sprayed with 103 EID50/mL or more of the virus. Based on half-life estimates, the feathers performed better than the corresponding media samples (p < 0.05), particularly when the samples were stored at 22 or 37 °C. In conclusion, feather samples can be routinely collected from a poultry barn as a non-invasive alternative to blood or oropharyngeal-cloacal swab samples for monitoring AIV.

The generation of hemagglutinin monoclonal antibodies against H9N2 influenza virus

H9N2 avian influenza viruses (AIVs) are widely distributed, causing continuous outbreaks in poultry and sporadic infections in humans. Vaccination is the primary method used to prevent and control H9N2 AIV infection. However, the ongoing evolution and mutation of AIVs often result in limited protection effects from vaccines. Therapeutic monoclonal antibodies (mAbs) targeting influenza viruses offer a promising alternative. In this study, we immunized mice with inactivated H9N2-W1 virus, and we screened and acquired five mAbs, namely 4D12, F4, 5C8, 2G8 and A11. We showed that all five mAbs specifically targeted the HA protein of various H9N2 AIV strains. In vitro neutralization tests demonstrated that all five mAbs exhibited neutralization activity against H9N2 AIVs, with mAb F4 displaying the most potent neutralization effect. The F4 mAb exhibited dose-dependent preventive and therapeutic effects against lethal H9N2-115 infection, and the administration of F4 at a dose of 3 μg/g provided complete protection in vivo. Our study presents an alternative approach for preventing and controlling H9N2 AIV infection. Furthermore, the identified F4 mAb holds promise as a solution to potential pandemics in humans caused by H9N2 AIVs.