Avian Influenza Virus Subtypes H5 Research Articles

Application of lentinan in suppression of Marek's disease virus infection

Marek's disease virus (MDV) is an extremely widespread avian immunosuppressive virus. In recent years, many reports have shown that there are still cases of MDV infection and immunosuppression after immunization with the vaccine. Consequently, there is a need to develop alternative complementary approaches for strengthening the efficacy of MDV prevention and control measures. Lentinan (LNT) is a macromolecular compound with immune-enhancing activity extracted from shiitake mushrooms. To explore the value and effectiveness of administering LNT through drinking water in the prevention and control of MDV, this study first observed the effects of high and low doses of LNT on weight gain, organ development, viral replication, and antibody titer of an avian influenza virus subtype H9 (AIV-H9) inactivated vaccine in specific pathogen-free (SPF) chicks infected with the wild strain of MDV. The results showed that both high and low doses of LNT significantly alleviated the weight gain retardation and liver and spleen enlargement caused by MDV infection, and significantly inhibited the replication of MDV in SPF chicks (P < 0.05), compared with the MDV-positive control group, both high and low doses of LNT significantly increased the antibody titer of AIV-H9 after immunization with inactivated AIV-H9 vaccine (P < 0.0001). On this basis, we also observed the effects of a chicken Marek's disease meq gene deletion live vaccine (SC9-1 strain), administered alone or in combination with LNT, on MDV infections of varying virulence in Hy-Line Brown chicks. The results showed that combined administration of LNT and the SC9-1 vaccine resulted in a significant alleviation of weight gain retardation and liver and spleen enlargement due to MDV infection (P < 0.05), as well as a significant inhibition of MDV replication and release in Hy-Line Brown chicks compared to the vaccine alone (P < 0.0001). These findings suggest that LNT not only alleviates the adverse effects of MDV infection in chickens but also enhances the efficacy of MDV vaccination, offering a potential auxiliary measure for controlling MDV infection.

Epidemiology and evolution of human-origin H10N5 influenza virus

H10 subtype avian influenza viruses were endemic in wild and domestic avian species worldwide. Strikingly, it frequently crossed the species barrier to infect mammalian hosts. Human infection with H10N3 and H10N8 were reported previously. Recently, a 63-year-old woman from Anhui province of China who died from a mixed infection of H3N2 and H10N5 influenza viruses, which have drawn widespread public health attention. Here, we perform the evolutionary dynamics of H10N5 influenza viruses of bird- and human-origin worldwide, and found that wild bird-origin H10N5 influenza viruses from China did not cluster together with human-origin H10N5 influenza viruses, while grouped together with LPAIV gene pools circulating in wild birds that derived from other Eurasian countries. Human-derived H10N5 virus is a novel reassortant, which frequently reassorted with wild bird-derived influenza viruses, and in turn, spillover into humans. Collectively, our results suggested that H10 subtype influenza viruses continuously pose threat to public health.

A Rapid Detection Method for H3 Avian Influenza Viruses Based on RT-RAA.

The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

An avian-origin internal backbone effectively increases the H5 subtype avian influenza vaccine candidate yield in both chicken embryonated eggs and MDCK cells

Inactivated vaccines play an important role in preventing and controlling the epidemic caused by the H5 subtype avian influenza virus. The vaccine strains are updated in response to alterations in surface protein antigens, while an avian-derived vaccine internal backbone with a high replicative capacity in chicken embryonated eggs and MDCK cells is essential for vaccine development. In this study, we constructed recombinant viruses using the clade 2.3.4.4d A/chicken/Jiangsu/GY5/2017(H5N6, CkG) strain as the surface protein donor and the clade 2.3.4.4b A/duck/Jiangsu/84512/2017(H5N6, Dk8) strain with high replicative ability as an internal donor. After optimization, the integration of the M gene from the CkG into the internal genes from Dk8 (8GM) was selected as the high-yield vaccine internal backbone, as the combination improved the hemagglutinin1/nucleoprotein (HA1/NP) ratio in recombinant viruses. The r8GMΔG with attenuated hemagglutinin and neuraminidase from the CkG exhibited high-growth capacity in both chicken embryos and MDCK cell cultures. The inactivated r8GMΔG vaccine candidate also induced a higher hemagglutination inhibition antibody titer and microneutralization titer than the vaccine strain using PR8 as the internal backbone. Further, the inactivated r8GMΔG vaccine candidate provided complete protection against wild-type strain challenge. Therefore, our study provides a high-yield, easy-to-cultivate candidate donor as an internal gene backbone for vaccine development.

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.

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

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

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.

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

Genetic Diversity and Biological Characteristics of H3 Avian Influenza Virus Isolated from China in 2021–2022 Showed the Emerging H3N8 Posed a Threat to Human Health

The H3 influenza viruses are widespread in domestic poultry but have been ignored because their pathogenicity in poultry is low. Three human infections with H3N8 influenza viruses have been reported in China since 2022, raising public concern. Here, we comprehensively analyzed 30 H3 subtype avian influenza viruses isolated from live poultry markets in China between 2021 and 2022. Genetic and phylogenetic analyses showed that the H3 viruses have undergone frequent reassortment and have formed complex genotypes. Notably, the viruses that caused human infections in 2022–2023 were highly homologous to the H3N8 viruses circulating in poultry in 2022, with internal genes derived from the H9N2 viruses. The analysis of chicken infections indicated that the novel H3N8 viruses were more infectious in chickens than those that do not carry H9N2 genes, whereas the H3 viruses detected in China in 2021–2022 showed low pathogenicity in mice. Our findings suggest that the novel H3N8 viruses bearing internal H9N2 genes have adapted to and circulated in chickens and pose a threat to human health. These results highlight the need for continued surveillance of the H3 influenza viruses and their impact on the poultry industry.

The adaptive potential of North American subtype H7N2 avian influenza viruses to mammals

Introduction. H7 subtype avian influenza viruses causing severe epizootics among birds are phylogenetically different in the Eastern and Western hemispheres. Numerous human infections caused by these viruses in the Eastern hemisphere indicate that H7 viruses can overcome the interspecies barrier and pose a potential threat of a new pandemic.The H7N2 viruses with deletion of amino acids 221–228 (H3 numbering) in hemagglutinin (HA) had been circulating among poultry in the Western Hemisphere during 1996–2006, and had once again been detected in 2016 in an animal shelter, where they caused cat diseases.&#x0D; The objective of this study is to elucidate the mechanism of adaptation to mammals of North American H7N2 influenza viruses with deletion in HA.&#x0D; Materials and methods. The A/chicken/New Jersey/294598-12/2004 (H7N2) virus was adapted to mice by the lung passages. Complete genomes of original and mouse-adapted viruses were analyzed. The receptor specificity and thermostability of viruses, HA activation pH and virulence for mice were determined.&#x0D; Results. The non-pathogenic H7N2 avian influenza virus became pathogenic after 10 passages in mice. Amino acid substitutions occurred in five viral proteins: one in PB2 (E627K), NA (K127N), NEP (E14Q), four in HA and six in NS1. Mutations in HA slightly changed receptor specificity but increased the pH of HA activation by 0.4 units. The NS1 protein undergone the greatest changes in the positions (N73T, S114G, K118R, G171A, F214L and G224R), where amino acid polymorphisms were observed in the original virus, but only minor amino acid variants have been preserved in the mouse adapted variant.&#x0D; Conclusion. The results show that H7N2 viruses have the potential to adapt to mammals. The increase in virulence is most likely due to the adaptive E627K mutation in PB2 and possibly in HA.

Expression and characterization of recombinant antibodies against H7 subtype avian influenza virus and their diagnostic potential.

Monoclonal antibodies (mAbs) play a pivotal role in disease diagnosis as well as immunotherapy interventions. Traditional monoclonal antibody generation relies on animal immunization procedures predominantly involving mice; however, recent advances in in-vitro expression methodologies have enabled large-scale production suitable for both industrial applications as well as scientific investigations. In this study, two mAbs against H7 subtype avian influenza viruses (AIV) were sequenced and analyzed, and the DNA sequences encoding heavy chain (HC) and light chain (LC) were obtained and cloned into pCHO-1.0 expression vector. Then, the HC and LC expression plasmids were transfected into CHO-S cells to establish stable cell lines expressing these mAbs using a two-phase selection scheme with different concentrations of methotrexate and puromycin. Recombinant antibodies were purified from the cell culture medium, and their potential applications were evaluated using hemagglutination inhibition (HI), western blotting (WB), confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results indicated that the obtained recombinant antibodies exhibited biological activity similar to that of the parent antibodies derived from ascites and could be used as a replacement for animal-derived mAbs. A kinetic analysis of the two antibodies to the AIV HA protein, conducted using surface plasmon resonance (SPR), showed concordance between the recombinant and parental antibodies. The data presented in this study suggest that the described antibody production protocol could avoid the use of experimental animals and better conform to animal welfare regulations, and provides a basis for further research and development of mAbs-based diagnostic products.

Concern regarding H3-subtype avian influenza virus.

The H3-subtype of avian influenza virus (AIV) is one of the most frequently detected low pathogenic avian influenza virus (LPAIV) subtypes in birds and fowls, causing substantial economic loss to the poultry industry. Most importantly, besides poultry, mammals could also be infected with it, such as swines, canines, equines, felines, and humans, posing a serious public health threat. This allows the virus to persist widely in poultry and wild birds for a long time, where it may mix with other subtypes, providing conditions for viral recombination or reassortment. Currently, the monitoring of H3-subtype AIV is inadequate, and there is a lack of effective prevention and control measures for H3-subtype AIV. Here, the epidemiology, phylogeny, and genetic variation of H3-subtype AIV were analyzed, and nonsynonymous and synonymous substitution rates (dN/dS) were calculated. Through these steps, we aimed to clarify the current epidemiological feature and evolutionary characteristics of H3-subtype AIV, and provide an operative reference for future scientific control of H3-subtype AIV.

Development and application of a triplex real-time PCR assay for the detection of H3, H4, and H5 subtypes of avian influenza virus

Avian influenza virus (AIV) poses a significant threat to the poultry industry and public health. Among the diverse AIV subtypes, H3, H4, and H5 are frequently detected in waterfowl and live poultry markets (LPM). The expeditious and precise identification of these subtypes is imperative in impeding the dissemination of the disease. In this study, we have developed a triplex real-time PCR assay endowed with the capacity to simultaneously discriminate AIV subtypes H3, H4, and H5. This method showcases remarkable specificity, selectively amplifying H3, H4, and H5 AIV subtypes sans any cross-reactivity with other subtypes or common avian pathogens. Furthermore, this method exhibits high sensitivity, with a detection threshold of 2.1 × 102 copies/μL for H3, H4, and H5 AIV subtypes. Additionally, the assay demonstrates reproducibility, as evidenced by intra- and inter-assay variability, with a coefficient of variation below 1.5%. A total of 338 cloacal swabs were collected from LPM to evaluate the performance of our assay. The obtained results evinced a high level of concordance with the sequencing data. In summary, our study has developed a triplex real-time PCR method that can be employed in laboratory-based testing and surveillance of AIV. This assay holds promise in augmenting our ability to detect and monitor AIV subtypes, thereby facilitating timely interventions and safeguarding both the poultry industry and public health.

Development of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid detection of H7 subtype avian influenza virus.

Avian influenza viruses (AIV) pose a significant persistent threat to the public health and safety. It is estimated that there have been over 100 outbreaks caused by various H7 subtypes of avian influenza viruses (AIV-H7) worldwide, resulting in over 33 million deaths of poultry. In this study, we developed a recombinase-aided amplification combined with a lateral flow dipstick assay for the detection of hemagglutinin (HA) genes to provide technical support for rapid clinical detection of AIV-H7. The results showed that the assay can complete the reaction within 30 min at a temperature of 39°C. Specificity tests demonstrated that there was no cross-reactivity with other common poultry pathogens, including Newcastle disease virus (NDV) and infections bronchitis virus (IBV). The detection limit of this assay was 1 × 101 copies/μL, while RT-qPCR method was 1 × 101 copies/μL, and RT-PCR was 1 × 102 copies/μL. The κ value of the RT-RAA-LFD and RT-PCR assay in 132 avian clinical samples was 0.9169 (p < 0.001). These results indicated that the developed RT-RAA-LFD assay had good specificity, sensitivity, stability and repeatability and may be used for rapid detection of AIV-H7 in clinical diagnosis.

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets.

Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus.

The avian influenza virus (AIV), a member of the type A influenza virus group, is harbored by avian hosts. The highly pathogenic H5 subtype of AIV has inflicted significant financial damage on the poultry industry and also presents a possible pandemic hazard to the worldwide human population. There is an urgent need for a detection method that is both rapid and accurate in identifying H5-AIV. In this study, two assays based on reverse transcription recombinase-aided amplification technology (RT-RAA) have been developed to detect H5-AIV, namely, real-time fluorescence and reverse transcription recombinase-aided amplification (RF-RT-RAA) and reverse transcription recombinase-aided amplification combined lateral flow dipstick (RT-RAA-LFD). The completion of the two methods can be achieved in 30 min, and there was no cross-reaction with the nucleic acid of other avian pathogens. The results indicated that the lowest detectable limit of RF-RT-RAA and RT-RAA-LFD for H5-AIV detection was 1 copy/μL, which was 100 times higher than that of RT-PCR (102 copies/μL). A total of 376 samples, consisting of 350 clinical and 26 experimental samples, underwent testing utilizing virus isolation, RF-RT-RAA and RT-RAA-LFD methods, respectively. The results demonstrated a high degree of consistency between those assays. In summary, both the RF-RT-RAA and RT-RAA-LFD methods demonstrated excellent specificity and sensitivity, making them ideal for use in both laboratory and field settings.IMPORTANCEAvian influenza virus (AIV) subtype H5 is a highly contagious zoonotic disease and a serious threat to the farming industry and public health. Traditional detection methods, including virus isolation and real-time PCR, require tertiary biological laboratories and are time-consuming and complex to perform, making it difficult to rapidly diagnose H5 subtype avian influenza viruses. In this study, we successfully developed two methods, namely, RF-RT-RAA and RT-RAA-LFD, for rapid detection of H5-AIV. The assays are characterized by their high specificity, sensitivity, and user-friendliness. Moreover, the results of the reaction can be visually assessed, which are suitable for both laboratory testing and grassroots farm screening for H5-AIV.

Genetic and biological properties of H10Nx influenza viruses in China

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.

The emergence of poultry-adapted H9 subtype avian influenza viruses. Proceeding of The First International Avian Influenza Summit, University of Arkansas- October 16-17, 2023"

Avian influenza is a highly contagious disease that severely affects poultry production and food security. Several countries have developed vaccination programs to prevent and control avian influenza. Unlike currently available vaccines, modified live virus (MLV) vaccines have the potential to be easily mass-applied. Concerns of reversion, recombination, and unintended transmission have slowed the development of MLVs against avian influenza for use in poultry. To address these concerns, we generated reassortment-impaired, non-transmissible, safe, immunogenic, and protective MLVs based on the rearrangement of internal gene segments and additional modifications of the surface gene segments HA and NA. The HA was produced to carry the unique DRPAVIAN peptide marker, whereas NA was modified as a chimera encoding the chicken interleukin-18 (ckIL18) gene (MLV-IL). As a proof of principle and given its economic importance and widespread geographic distribution, we tested the MLV and MLV-IL approaches in chickens against a prototypic H9N2 low pathogenic avian influenza virus (MLV-H9N2 and MLV-H9N2-IL, respectively). \textit{In-vitro}, the MLV-H9N2 and MLV-H9N2-IL candidates were stable and achieved yields similar to the wild-type H9N2 strain. Co-infection studies with a wild-type H9N2 virus showed that the altered HA segment in the MLV-H9N2 and MLV-H9N2-IL candidates has a fitness disadvantage and did not reassort. Likewise, the modified NA segment in the MLV-H9N2-IL candidate was impaired in reassortment. \textit{In-vivo}, the MLV-H9N2 and MLV-H9N2-IL candidates did not transmit chicken-to-chicken via direct contact. MLV-H9N2- and MLV-H9N2-IL-vaccinated chickens showed no clinical signs upon intranasal vaccination, and all were seroconverted. More importantly, including ckIL18 in the MLV-H9N2-IL vaccine enhanced the generation of neutralizing antibodies, suggesting a role in the host immune response. A significant decrease in viral loads post-challenge demonstrated the protective effect of the MLVs. The MLV-H9N2-IL vaccine administered via drinking water was immunogenic in chickens in a dose-dependent manner and generated levels of neutralizing antibodies after prime and boost predictive of protection, which was later confirmed upon aggressive homologous virus challenge. In summary, this work provides the basis for novel and safe MLVs against avian influenza amenable for mass vaccination.

Virulence and transmission characteristics of clade 2.3.4.4b H5N6 subtype avian influenza viruses possessing different internal gene constellations

ABSTRACT Clade 2.3.4.4 H5N6 avian influenza virus (AIV) has been predominant in poultry in China, and the circulating haemagglutinin (HA) gene has changed from clade 2.3.4.4h to clade 2.3.4.4b in recent years. In 2021, we isolated four H5N6 viruses from ducks during the routine surveillance of AIV in China. The whole-genome sequencing results demonstrated that the four isolates all belonged to the currently prevalent clade 2.3.4.4b but had different internal gene constellations, which could be divided into G1 and G2 genotypes. Specifically, G1 possessed H9-like PB2 and PB1 genes on the H5-like genetic backbone while G2 owned an H3-like PB1 gene and the H5-like remaining internal genes. By determining the characteristics of H5N6 viruses, including growth performance on different cells, plaque-formation ability, virus attachment ability, and pathogenicity and transmission in different animal models, we found that G1 strains were more conducive to replication in mammalian cells (MDCK and A549) and BALB/c mice than G2 strains. However, G2 strains were more advantageously replicated in avian cells (CEF and DF-1) and slightly more transmissible in waterfowls (mallards) than G1 strains. This study enriched the epidemiological data of H5 subtype AIV to further understand its dynamic evolution, and laid the foundation for further research on the mechanism of low pathogenic AIV internal genes in generating novel H5 subtype reassortants.