HA Gene Research Articles

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus.

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.

Epidemiological and Genetic Characterization of Human Infection with Avian Influenza AI H5N6 Virus in Guangxi, China, 2021

Avian influenza (AI) H5N6 has replaced H5N1 as the predominant strain, and the increasing number of cases and complex recombination patterns pose significant threats to human health. The data were collected on human cases of AI H5N6 from the National Surveillance of Notifiable Infectious Disease Programme (NSNIDP) in Guangxi and used MEGA software to conduct phylogenetic analysis with published reference strains obtained from the GISAID database. Between January 1 and December 31, 2021, 11 cases of human AI H5N6 infection were reported in Guangxi. All HA genes belonged to clade 2.3.4.4, with only one virus belonging to 2.3.4.4h and the remaining viruses falling into 2.3.4.4b. Phylogenetic analysis of the genes was conducted, and some substitutions were detected in the HA gene, M1 protein, and NS1 protein. A close relationship between Guangxi viruses and AIVs of poultry origin has been observed. Given the contamination of live poultry markets and backyard poultry, AI H5N6 variants and genotypes will continuously emerge; therefore, enhancing surveillance is crucial for pandemic preparedness.

Insights into Genetic and Antigenic Characteristics of Influenza A(H1N1)pdm09 Viruses Circulating in Sicily During the Surveillance Season 2023-2024: The Potential Effect on the Seasonal Vaccine Effectiveness.

After disruption in the influenza circulation due to the emergence of SARS-CoV-2, the intensity of seasonal outbreaks has returned to the pre-pandemic levels. This study aimed to evaluate the evolution and variability of whole-genome sequences of A(H1N1)pdm09, the predominant influenza virus in Sicily (Italy) during the season 2023-2024. The potential vaccine efficacy was calculated using the pepitope model based on amino acid changes in the dominant epitope of hemagglutinin. The HA gene sequences showed several amino acid substitutions, some of which were within the major antigenic sites. The phylogenetic analysis showed that Sicilian strains grouped into two main genetic clades (6B.1A.5a.2a.1 and 6B.1A.5a.2a) and several subclades. Notably, about 40% of sequences partially drifted from the WHO-recommended vaccine strain A/Victoria/4897/2022 for the Northern Hemisphere. These sequences mostly belonged to the subclades C.1.8 and C.1.9 and harboured the amino acid mutations responsible for the modest predicted vaccine efficacy (E = 38.12% of 53%, pepitope = 0) against these viruses. Amino acid substitutions in other gene segments were also found. Since influenza viruses are constantly evolving, genomic surveillance is crucial in monitoring their molecular evolution and the occurrence of genetic and antigenic changes, and, thus, their potential impact on vaccine efficacy.

Epidemiology and biological characteristics of influenza A (H4N6) viruses from wild birds

ABSTRACT During the active surveillance, we isolated nine H4N6 subtype influenza A viruses from wild birds in China. To reveal the epidemiology and biology characteristics of H4 subtype influenza A virus from wild birds, we investigated H4 subtype viruses available in the public source, and found that the H4 viruses have been detected in at least 37 countries to date, and more than 73.6% of the viruses were from wild Anseriformes. Bayesian phylogeographic analysis showed that Mongolia worked as the important transmission centre for Eurasian lineage H4 viruses spreading. Phylogenetic analysis of HA genes indicated that global H4 influenza A viruses were divided into Eurasian and North American lineage, our nine H4N6 isolates fell into the Eurasian lineage. Recombination analysis suggested that nine H4N6 isolates underwent complex gene recombination with various subtypes of influenza A viruses and formed two genotypes. Notably, nine H4N6 isolates acquired mammalian virulence-increasing residues. Two representative H4N6 viruses possessed dual receptor binding specificity, they could efficiently replicate in MDCK and 293 T cells in vitro infection, also could cross the species barrier to infect mice directly without prior adaption in vivo experiments. These findings emphasize the public health issues represented by H4 viruses, and highlight the need to strengthen the active surveillance of H4 viruses from wild birds.

Genomic Surveillance and Evolutionary Dynamics of Influenza A Virus in Sri Lanka.

Background Influenza A has been named as a priority pathogen by the WHO due to the potential to cause pandemics. Genomic sequencing of influenza strains is important to understand the evolution of the influenza strains and also to select the appropriate influenza vaccines to be used in the different influenza seasons in Sri Lanka. Therefore, we sought to understand the molecular epidemiology of the influenza viruses in the Western Province of Sri Lanka, including mutational analysis to investigate the evolutionary dynamics. Methods A total of 349 individuals presenting with fever and respiratory symptoms were enrolled in this study from November 2022 to May 2024. Nasopharyngeal and oropharyngeal specimens were collected and screened using quantitative PCR to detect Influenza A, Influenza B, and SARS-CoV-2. Subtyping and genomic sequencing was carried out on influenza A strains using Oxford Nanopore Technology. Results Influenza A was detected in 49 (14%) patients, influenza B in 20 (5.7%) and SARS-CoV-2 in 41 (11.7%). Co-infections were observed in five participants. The phylogenetic analysis assigned the H1N1 HA gene sequences within the 6B.1A.5a.2a clade. The HA gene of the H1N1 sequences in 2023 were assigned as belonging to the subclades C.1, C.1.2, and C.1.8, while the 2024 sequences were assigned to subclades C.1.8 and C.1.9. The H3N2 sequences from 2023 were assigned to the 3C.2a1b.2a.2a.1b clade and subclade G.1.1.2, while the 2024 sequences were assigned to the 3C.2a1b.2a.2a.3a.1 clade and subclade J.2. The K54Q, A186T, Q189E, E224A, R259K, K308R, I418V, and X215A amino acid substitutions were seen in the H1N1 in the 2023 and 2024 sequences. The 2024 H1N1 sequences additionally exhibited further substitutions, such as V47I, I96T, T120A, A139D, G339X, K156X, and T278S. Conclusion In this first study using genomic sequencing to characterize the influenza A strains in Sri Lanka, which showed different influenza A viruses circulating in an 18-month period. As the Sri Lankan strains also had certain mutations of unknown significance, it would be important to continue detailed surveillance of the influenza strains in Sri Lanka to choose the most suitable vaccines for the population and the timing of vaccine administration.

Molecular epidemiology of avian influenza viruses and avian coronaviruses in environmental samples from migratory bird inhabitants in Bangladesh.

Migratory birds are a natural reservoir for major respiratory viruses such as the avian influenza virus (AIV) and the avian coronavirus (AvCoV). Transmission of these viruses from migratory birds to domestic birds increases the prevalence of those diseases that cause severe economic and public health concerns in Bangladesh. The study focused on active surveillance of major respiratory viral pathogens in migratory birds, molecular identification of the viruses, and their phylogenetic origin. To conduct this study, 850 environmental samples (830 fecal samples, 10 soil samples, and 10 water samples) were collected during three consecutive winter seasons from three divisions (Dhaka, Sylhet, and Mymensingh) and pooled according to the year of collection and locations, resulting in a total of 184 tested samples. Using gene-specific primers and probes in TaqMan-and SYBR Green-based RT-qPCR assays, the samples were screened for AIV and AvCoV, respectively. Out of the 184 pooled samples, 37 were found to be positive for these respiratory pathogens. Furthermore, out of the 37 (20.11%) positive respiratory pathogens, 11.96% were AIV (n = 22) and 8.15% were AvCoV (n = 15). For the first time in Bangladesh, AIV H4N2, H4N6, and AvCoVs have been found in fecal samples from migratory birds through surveillance. Phylogenetic analyses of the HA and NA genes of AIV and the polymerase gene (Orf 1) of AvCoV revealed that these strains share a close phylogenetic relationship with the isolates from wild birds in Europe and Asia. The Bangladeshi strains with Eurasian ancestry might pose a significant threat to migratory birds flying through the Asian flyways. They might also be a potential source of virus introduction and spread to poultry raised on land. These findings emphasize the significance of ongoing AIV and AvCoV surveillance in migratory birds in Bangladesh.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

The evolutionary features and roles of single nucleotide variants and charged amino acid mutations in influenza outbreaks during NPI period

The epidemic and outbreaks of influenza B Victoria lineage (Bv) during 2019–2022 led to an analysis of genetic, epitopes, charged amino acids and Bv outbreaks. Based on the National Influenza Surveillance Network (NISN), the Bv 72 strains isolated during 2019–2022 were selected by spatio-temporal sampling, then were sequenced. Using the Compare Means, Correlate and Cluster, the outbreak data were analyzed, including the single nucleotide variant (SNV), amino acid (AA), epitope, evolutionary rate (ER), Shannon entropy value (SV), charged amino acid and outbreak. With the emergence of COVID-19, the non-pharmaceutical interventions (NPIs) made Less distant transmission and only Bv outbreak. The 2021–2022 strains in the HA genes were located in the same subset, but were distinct from the 2019–2020 strains (P < 0.001). The codon G → A transition in nucleotide was in the highest ratio but the transversion of C → A and T → A made the most significant contribution to the outbreaks, while the increase in amino acid mutations characterized by polar, acidic and basic signatures played a key role in the Bv epidemic in 2021–2022. Both ER and SV were positively correlated in HA genes (R = 0.690) and NA genes (R = 0.711), respectively, however, the number of mutations in the HA genes was 1.59 times higher than that of the NA gene (2.15/1.36) from the beginning of 2020 to 2022. The positively selective sites 174, 199, 214 and 563 in HA genes and the sites 73 and 384 in NA genes were evolutionarily selected in the 2021–2022 influenza outbreaks. Overall, the prevalent factors related to 2021–2022 influenza outbreaks included epidemic timing, Tv, Ts, Tv/Ts, P137 (B → P), P148 (B → P), P199 (P → A), P212 (P → A), P214 (H → P) and P563 (B → P). The preference of amino acid mutations for charge/pH could influence the epidemic/outbreak trends of infectious diseases. Here was a good model of the evolution of infectious disease pathogens. This study, on account of further exploration of virology, genetics, bioinformatics and outbreak information, might facilitate further understanding of their deep interaction mechanisms in the spread of infectious diseases.

Genomic Surveillance and Evolutionary Dynamics of Influenza A Virus in Sri Lanka.

Influenza A has been named as a priority pathogen by the WHO due to the potential to cause pandemics. Genomic sequencing of influenza strains is important to understand the evolution of the influenza strains and also to select the appropriate influenza vaccines to be used in the different influenza seasons in Sri Lanka. Therefore, we sought to understand the molecular epidemiology of the influenza viruses in the Western Province of Sri Lanka, including mutational analysis to investigate the evolutionary dynamics. A total of 349 individuals presenting with fever and respiratory symptoms were enrolled in this study from November 2022 to May 2024. Nasopharyngeal and oropharyngeal specimens were collected and screened using quantitative PCR to detect Influenza A, Influenza B, and SARS-CoV-2. Subtyping and genomic sequencing was carried out on influenza A strains using Oxford Nanopore Technology. Influenza A was detected in 49 (14 %) patients, influenza B in 20 (5.7%) and SARS-CoV-2 in 41 (11.7%). Co-infections were observed in five participants. The phylogenetic analysis assigned the H1N1 HA gene sequences within the 6B.1A.5a.2a clade. The HA gene of the H1N1 sequences in 2023 were assigned as belonging to the subclades C.1, C.1.2, and C.1.8, while the 2024 sequences were assigned to subclades C.1.8 and C.1.9. The H3N2 sequences from 2023 were assigned to the 3C.2a1b.2a.2a.1b clade and subclade G.1.1.2, while the 2024 sequences were assigned to the 3C.2a1b.2a.2a.3a.1 clade and subclade J.2. The K54Q, A186T, Q189E, E224A, R259K, K308R, I418V, and X215A amino acid substitutions were seen in the H1N1 in the 2023 and 2024 sequences. The 2024 H1N1 sequences additionally exhibited further substitutions, such as V47I, I96T, T120A, A139D, G339X, K156X, and T278S. In this first study using genomic sequencing to characterize the influenza A strains in Sri Lanka, which showed different influenza A viruses circulating in an 18-month period. As the Sri Lankan strains also had certain mutations of unknown significance, it would be important to continue detailed surveillance of the influenza strains in Sri Lanka to choose the most suitable vaccines for the population and the timing of vaccine administration.

A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes

The global spread of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses threatens poultry and public health. The continuous circulation of these viruses has led to their considerable genetic and antigenic evolution, resulting in the formation of eight subclades (2.3.4.4a–h). Here, we examined the antigenic sites that determine the antigenic differences between two H5 vaccine strains, H5-Re8 (clade 2.3.4.4g) and H5-Re11 (clade 2.3.4.4h). Epitope mapping data revealed that all eight identified antigenic sites were located within two classical antigenic regions, with five sites in region A (positions 115, 120, 124, 126, and 140) and three in region B (positions 151, 156, and 185). Through antigenic cartography analysis of mutants with varying numbers of substitutions, we confirmed that a combination of mutations in these eight sites reverses the antigenicity of H5-Re11 to that of H5-Re8, and vice versa. More importantly, our analyses identified H5-Re11_Q115L/R120S/A156T (H5-Re11 + 3) as a promising candidate for a broad-spectrum vaccine, positioned centrally in the antigenic map, and offering potential universal protection against all variants within the clade 2.3.4.4. H5-Re11 + 3 serum has better cross-reactivity than sera generated with other 2.3.4.4 vaccines, and H5-Re11 + 3 vaccine provided 100% protection of chickens against antigenically drifted H5 viruses from various 2.3.4.4 antigenic groups. Our findings suggest that antigenic regions A and B are immunodominant in H5 viruses, and that antigenic cartography-guided vaccine design is a promising strategy for selecting a broad-spectrum vaccine.

Detection and typing of influenza viruses in vaccinated and non- vaccinated children in central Greece during 2018-2019

During the 2018-2019 influenza season in central Greece, 388 respiratory samples were collected from children presented with influenza symptoms and tested for the detection of influenza viruses. Real-time RT-PCR testing of the samples revealed the presence of influenza A (H1N1) (pdm09), influenza A (H3N2), seasonal influenza A (H1N1), and influenza B viruses. All samples tested were found negative for influenza A(H5) HA gene sequence. The majority of influenza viruses detected were type A (90%), of which the majority, (61%), were influenza A (H1N1) (pdm09) viruses, indicating that these viruses were the dominant influenza subtype circulating in central Greece during the winter and early spring of the aforementioned season.

Effects of different HA and NA gene combinations on the growth characteristics of the H3N8 influenza candidate vaccine virus

Since 2022, three human cases of a novel H3N8 avian influenza virus infection have been reported in three provinces in China. Specific vaccines are important means of preparing for the potential influenza pandemic. Thus, H3N8 viruses [A/Henan/cnic410/2022 (HN410) and A/Changsha/1000/2022(CS1000)] were isolated from the infected patients as prototype viruses to develop candidate vaccine viruses (CVVs) using the reverse genetics (RG) technology. Five reassortant viruses with different HA and NA combinations were constructed based on the two viruses to get a high-yield and safe CVV. The results showed that all viruses had similar antigenicity but different growth characteristics. Reassortant viruses carrying NA from CS1000 exhibited better growth ability and NA enzyme activity than the ones carrying HN410 NA. Furthermore, the NA gene of CS1000 had one more potential N-glycosylation site at position 46 compared with HN410. The substitution of position 46 showed that adding or removing N-glycosylation sites to different reassortant viruses had different effects on growth ability. A reassortant virus carrying HN410 HA and CS1000 NA with high growth ability was selected as a CVV, which met the requirements for a CVV. These data suggest that different surface gene combinations and the presence or absence of potential N-glycosylation sites on position 46 in the NA gene affect the growth characteristics of H3N8 CVVs.

Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle

ABSTRACT In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Genetic Reassortment in a Child Coinfected with Two Influenza B Viruses, B/Yamagata Lineage and B/Victoria-Lineage Strains.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Insect Cell-Based Quadrivalent Seasonal Influenza Virus-like Particles Vaccine Elicits Potent Immune Responses in Mice.

Influenza viruses can cause highly infectious respiratory diseases, posing noteworthy epidemic and pandemic threats. Vaccination is the most cost-effective intervention to prevent influenza and its complications. However, reliance on embryonic chicken eggs for commercial influenza vaccine production presents potential risks, including reductions in efficacy due to HA gene mutations and supply delays due to scalability challenges. Thus, alternative platforms are needed urgently to replace egg-based methods and efficiently meet the increasing demand for vaccines. In this study, we employed a baculovirus expression vector system to engineer HA, NA, and M1 genes from seasonal influenza strains A/H1N1, A/H3N2, B/Yamagata, and B/Victoria, generating virus-like particle (VLP) vaccine antigens, H1N1-VLP, H3N2-VLP, Yamagata-VLP, and Victoria-VLP. We then assessed their functional and antigenic characteristics, including hemagglutination assay, protein composition, morphology, stability, and immunogenicity. We found that recombinant VLPs displayed functional activity, resembling influenza virions in morphology and size while maintaining structural integrity. Comparative immunogenicity assessments in mice showed that our quadrivalent VLPs were consistent in inducing hemagglutination inhibition and neutralizing antibody titers against homologous viruses compared to both commercial recombinant HA and egg-based vaccines (Vaxigrip). The findings highlight insect cell-based VLP vaccines as promising candidates for quadrivalent seasonal influenza vaccines. Further studies are worth conducting.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

N-glycosylation on hemagglutinin head reveals inter-branch antigenic variability of avian influenza virus H5-subtypes

H5-subtype avian influenza virus (AIV) is globally prevalent and undergoes frequent antigenic drift, necessitating regular updates to vaccines. One of the many influencing elements that cause incompatibility between vaccinations and epidemic strains is the dynamic alteration of glycosylation sites. However, the biological significance of N-glycosylation in the viral evolution and antigenic changes is unclear. Here, we performed a systematic analysis of glycosylation sites on the HA1 subunit of H5N1, providing insights into the changes of primary glycosylation sites, including 140 N, 156 N, and 170 N within the antigenic epitopes of HA1 protein. Multiple recombinant viruses were then generated based on HA genes of historical vaccine strains and deactivated for immunizing SPF chickens. Inactivated recombinant strains showed relatively closer antigenicity compared to which has identical N-glycosylation patterns. The N-glycosylation modification discrepancy highlights the inter-branch antigenic diversity of H5-subtype viruses in avian influenza and serves as a vital foundation for improving vaccination tactics.

Pathogenicity and transmission of novel highly pathogenic H7N2 variants originating from H7N9 avian influenza viruses in chickens

The H7 subtype avian influenza viruses are circulating widely worldwide, causing significant economic losses to the poultry industry and posing a serious threat to human health. In 2019, H7N2 and H7N9 co-circulated in Chinese poultry, yet the risk of H7N2 remained unclear. We isolated and sequenced four H7N2 viruses from chickens, revealing them as novel reassortants with H7N9-derived HA, M, NS genes and H9N2-derived PB2, PB1, PA,NP, NA genes. To further explore the key segment of pathogenicity, H7N2-H7N9NA and H7N2-H9N2HA single-substitution were constructed. Pathogenicity study showed H7N2 isolates to be highly pathogenic in chickens, with H7N2-H7N9NA slightly weaker than H7N2-Wild type. Transcriptomic analysis suggested that H7N9-derived HA genes primarily drove the high pathogenicity of H7N2 isolates, eliciting a strong inflammatory response. These findings underscored the increased threat posed by reassorted H7N2 viruses to chickens, emphasizing the necessity of long-term monitoring of H7 subtype avian influenza viruses.

Phylogeography and gene pool analysis of highly pathogenic avian influenza H5N1 viruses reported in India from 2006 to 2021.

India has reported highly pathogenic avian influenza (HPAI) H5N1 virus outbreaks since 2006, with the first human case reported in 2021. These included viruses belonging to the clades 2.2, 2.2.2, 2.2.2.1, 2.3.2.1a, and 2.3.2.1c. There are currently no data on the gene pool of HPAI H5N1 viruses in India. Molecular clock and phylogeography analysis of the HA and NA genes; and phylogenetic analysis of the internal genes of H5N1 viruses from India were carried out. Sequences reported from 2006 to 2015; and sequences from 2021 that were available in online databases were used in the analysis. Five separate introductions of H5N1 viruses into India were observed, via IndonesiaorKorea (2002), Bangladesh (2009), Bhutan (2010), and China (2013, 2018) (clades 2.2, 2.2.2, 2.2.2.1, 2.3.2.1a, 2.3.2.1c, and 2.3.4.4b). Phylogenetic analysis revealed eight reassortant genotypes. The H5N1 virus isolated from the human case showed a unique reassortant genotype. Amino acid markers associated with adaptation to mammals were also present. This is the first report of the spatio-temporal origins and gene pool analysis of H5N1 viruses from India, highlighting the need for increased molecular surveillance.

Molecular Characterization of Non-H5 and Non-H7 Avian Influenza Viruses from Non-Mallard Migratory Waterbirds of the North American Flyways, 2006-2011.

The surveillance of migratory waterbirds (MWs) for avian influenza virus (AIV) is indispensable for the early detection of a potential AIV incursion into poultry. Surveying AIV infections and virus subtypes in understudied MW species could elucidate their role in AIV ecology. Oropharyngeal-cloacal (OPC) swabs were collected from non-mallard MWs between 2006 and 2011. OPC swabs (n = 1158) that molecularly tested positive for AIV (Cts ≤ 32) but tested negative for H5 and H7 subtypes were selected for virus isolation (VI). The selected samples evenly represented birds from all four North American flyways (Pacific, Central, Mississippi, and Atlantic). Eighty-seven low pathogenic AIV isolates, representing 31 sites in 17 states, were recovered from the samples. All isolates belonged to the North American lineage. The samples representing birds from the Central Flyway had the highest VI positive rate (57.5%) compared to those from the other flyways (10.3-17.2%), suggesting that future surveillance can focus on the Central Flyway. Of the isolates, 43.7%, 12.6%, and 10.3% were obtained from blue-winged teal, American wigeon, and American black duck species, respectively. Hatch-year MWs represented the majority of the isolates (70.1%). The most common H and N combinations were H3N8 (23.0%), H4N6 (18.4%), and H4N8 (18.4%). The HA gene between non-mallard and mallard MW isolates during the same time period shared 85.5-99.5% H3 identity and 89.3-99.7% H4 identity. Comparisons between MW (mallard and non-mallard) and poultry H3 and H4 isolates also revealed high similarity (79.0-99.0% and 88.7-98.4%), emphasizing the need for continued AIV surveillance in MWs.