The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination.

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

The haemagglutinin-neuraminidase protein of velogenic Newcastle disease virus enhances viral infection through NF-κB-mediated programmed cell death.

The haemagglutinin-neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.

Amino acid mutations PB1-V719M and PA-N444D combined with PB2-627K contribute to the pathogenicity of H7N9 in mice.

H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein.

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses.

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

G1-like PB2 gene improves virus replication and competitive advantage of H9N2 virus.

H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007 with the substitution of F98-like PB2 and M gene by G1-like ones, has become the overwhelming predominant genotype for the past 11 years since 2010. Here, we found that virus with G1-like PB2 were more efficient in protein expression and in infectious virus production than that with F98-like PB2 gene. By coinfected MDCK cells with the reassortant virus, more survival opportunity for viruses with G1-like PB2 than that of F/98-like was observed. Besides, in animal experiments, we found that the G1-like PB2 increases virus infectivity, replication, and virus shedding of H9N2 in chickens. Our results suggested that the substitution of G1-like PB2 play important role in promoting the fitness of genotype S H9N2 virus in China.

Highly-expressed micoRNA-21 in adipose derived stem cell exosomes can enhance the migration and proliferation of the HaCaT cells by increasing the MMP-9 expression through the PI3K/AKT pathway.

OBJECTIVE: Wound healing remains a challenge in burns and trauma fields. Adipose derived stem cells exosomes (AD-exos) had been confirmed to have a positive effect on the wound healing and the migration and proliferation of keratinocyte. However, the mechanism of the AD-exos is still unclear. The objective of this article is to observe the function of the miR-21 expressed in the adipose AD-exos and the effect on migration and proliferation of the HaCaT cells. MATERIALS AND METHODS: The full layer dermal wound of BALb/c mouse was used to observe the vitro effect of the AD-exos and detect the expression of miR-21.The co-culture systems were established by transwell plates for observing the migration, proliferation, apoptosis rate, detecting the RNA, and protein expression in different treated groups. MiR-21 plasmid was used to over-express miR-21 by transfection of HaCaT cells. GW4869 was used to inhibit the secreting of exosomes from ADSCs. RESULTS: The results showed that both ADSCs and the AD-exos could improve the wound healing process of BALb/c mouse full layer skin wound at a similar level, especially at the 7th day post surgery when compared to the control group (p < 0.01) and the highly expressed miR-21 was detected (p < 0.01 compared with control group and p < 0.001 compared to other microRNAs) in the treated groups at the same time point. AD-exos could obviously enhance the migration and proliferation of the HaCaT cells (p < 0.01), and fell back to the same level when the exosomes inhibitor--GW4869 was added compared with control group (p > 0.05). Over-expressed miR-21 could also significantly improve the migration and proliferation of HaCaT cells. But both AD-exos and miR-21 had no significantly effect on the apoptosis rate of HaCaT cells (p > 0.05 compared with each other). Over-expression of miR-21 plasmid could decrease the TGF-ßI protein level (p < 0.001 vs. control group) in HaCaT cells while TGF-ßI protein level increased again when antagomiR-21 was added in (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 expression of HaCaT cells could be increased by the transfect ion of miR-21 plasmid (p < 0.001 vs. empty plasmid group) and decreased by antagomiR-21 (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 appeared to have influence on MMP-9 and TIMP-2 (p < 0.001 compared to control group and p < 0.001 compared to TGF-ßI group) but not MMP-2 and TIMP-1 (p > 0.05 compared to control group and TGF-ßI group). These processes might act through PI3K/AKT pathway. CONCLUSION: This research provide the experimental evidence that the miR-21 is highly expressed in AD-exos and can significantly accelerate the wound healing process and enhance the migration and proliferation of the HaCaT cells. Over-expressed miR-21 can inhibit the TGF-ßI expression and excess TGF-ßI can also have negative feedback influence on miR-21. We have found a reliable evidence that these two factors can act on HaCaT cells by influencing MMP-2 and TIMP-1 protein expression through the PI3K/AKT signal pathway. These results may provide a potential perspectives on improving the wound healing.

Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus.

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.

The PA-interacting host protein nucleolin acts as an antiviral factor during highly pathogenic H5N1 avian influenza virus infection.

Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-ß, CCL-4, CCL-8, IFN-α, IFN-ß and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses.

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 103.0EID50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Phylogenetic and biological characterization of three K1203 (H5N8)-like avian influenza A virus reassortants in China in 2014.

Three H5N8 avian influenza viruses isolated from domestic geese in China in 2014 were characterized phylogenetically and biologically. Phylogenetic analysis of the complete genomic sequences of the three isolates from this study and those of 61 other H5N8 viruses retrieved from the GISAID platform indicated that, chronologically and geographically, all H5N8 viruses of the Asian H5N1 HA lineage of clade 2.3.4.4 are the direct descendents of the K1203 (H5N8)-like viruses first isolated in China in 2010. The three viruses from this study shared high sequence similarity in all eight gene segments with three other isolates from China in 2013, and two Korean isolates were distinct from the recently circulating reassortants causing outbreaks in Asia, Europe and the United States in 2014 and 2015. In vitro viral growth curves indicated that these H5N8 viruses replicated to high titers in CEF, DEF, MDCK and A549 cells but to significantly lower titers in Vero cells. Pathogenicity studies in vivo indicated that these viruses were all highly virulent to chickens and mallard ducks, while they varied from moderate to high virulence in mice. Additionally, hemagglutination assays using α-2,3-sialidase-treated goose red blood cells and solid-phase direct binding assays with different glycans demonstrated that the three viruses could bind to both avian-type SAα-2,3Gal and human-type SAα-2,6Gal receptors. Our findings confirmed the progenitor nature of the K1203-like viruses in generating recent prevalent clade 2.3.4.4 H5N8 reassortants, which have caused tremendous damage to the poultry industry and are a potential threat to public health.

Influenza A virus infection activates STAT3 to enhance SREBP2 expression, cholesterol biosynthesis, and virus replication.

Cellular cholesterol plays an important role in influenza A virus (IAV) endocytosis and replication. However, how IAV infection regulates cholesterol biosynthesis remains poorly understood. Here, we report that IAV infection activates SREBP2 and induces the expression of HMGCR, a rate-limiting enzyme in cholesterol synthesis pathway. SREBP2 deficiency suppresses IAV-induced HMGCR expression and virus replication. Mechanistically, IAV infection activates JAK2 and STAT3, inhibition of JAK2 and STAT3 activity by their inhibitors or by gene knockout downregulates IAV-induced SREBP2 and HMGCR expression and IAV replication, reduces the content of cellular cholesterol and virus binding to host cells. Exogenous cholesterol reverses the inhibitory effect of S3I-201 and STAT3 deficiency on virus replication. STAT3 or JAK2 overexpression increases the expression of SREBP2 and its downstream target genes, leading to increased IAV replication. These observations collectively suggest that STAT3 activation facilitates IAV replication by inducing SREBP2 expression and increasing cholesterol biosynthesis.

Mesenchymal stem cells prevent H7N9 virus infection via rejuvenating immune environment to inhibit immune-overactivity.

BACKGROUND: Influenza is a clinically important infectious disease with a high fatality rate, which always results in severe pneumonia. Mesenchymal stem cells (MSCs) exhibit promising therapeutic effects on severe viral pneumonia, but whether MSCs prevent virus infection and contribute to the prevention of influenza remains unknown. METHODS: ICR mice were pretreated with human umbilical cord (hUC) MSCs and then infected with the influenza H7N9 virus. Weight, survival days, and lung index of mice were recorded. Serum antibody against influenza H7N9 virus was detected according to the hemagglutination inhibition method. Before and after virus infection, T cell and B cell subtypes in the peripheral blood of mice were evaluated by flow cytometry. Cytokines in the supernatants of MSCs, innate immune cells, and mouse broncho alveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA) or Luminex Assay. RESULTS: Pretreatment with MSCs protected mice against influenza H7N9 virus infection. Weight loss, survival rate, and structural and functional damage to the lungs of infected mice were significantly improved. Mechanistically, MSCs modulated T lymphocyte response in virus-infected mice and inhibited the cGAS/STING pathway. Importantly, the protective effect of MSCs was mediated by cell-to-cell communications and attenuation of cytokine storm caused by immune overactivation.

Hemagglutinin affects replication, stability and airborne transmission of the H9N2 subtype avian influenza virus.

H9N2 subtype avian influenza virus (AIV) can transmit by direct as well as airborne contacts. It has been widespread in poultry and continued to contribute to zoonotic spillover events by providing its six internal genes for the reassortment of novel influenza viruses (eg, H7N9) that infect poultry and humans. Compared to H7N9, H9N2 virus displays an efficient airborne transmissibility in poultry, but the mechanisms of transmission difference have been insufficiently studied. The Hemagglutinin (HA) and viral polymerase acidic protein (PA) have been implicated in the airborne transmission of influenza A viruses. Accordingly, we generated the reassortant viruses of circulating airborne transmissible H9N2 and non-airborne transmissible H7N9 viruses carrying HA and/or PA gene. The introduction of the PA gene from H7N9 into the genome of H9N2 virus resulted in a reduction in airborne transmission among chickens, while the isolated introduction of the HA gene segment completely eliminated airborne transmission among chickens. We further showed that introduction of HA gene of non-transmissible H7N9 did not influence the HA/NA balance of H9N2 virus, but increased the threshold for membrane fusion and decreased the acid stability. Thus, our results indicate that HA protein plays a key role in replication, stability, and airborne transmission of the H9N2 subtype AIV.

Imaging feature-based clustering of financial time series.

Timeseries representation underpin our ability to understand and predict the change of natural system. Series are often predicated on our choice of highly redundant factors, and in fact, the system is driven by a much smaller set of latent intrinsic keys. It means that a better representation of data makes points in phase space clearly for researchers. Specially, a 2D structure of timeseries could combine the trend and correlation characters of different periods in timeseries together, which provides more clear information for top tasks. In this work, the effectiveness of 2D structure of timeseries is investigated in clustering tasks. There are 4 kinds of methods that the Recurrent Plot (RP), the Gramian Angular Summation Field (GASF), the Gramian Angular Differential Field (GADF) and the Markov Transition Field (MTF) have been adopted in the analysis. By classifying the CSI300 and S&P500 indexes, we found that the RP imaging series are valid in recognizing abnormal fluctuations of financial timeseries, as the silhouette values of clusters are over 0.6 to 1. Compared with segment methods, the 2D models have the lowest instability value of 0. It verifies that the SIFT features of RP images take advantage of the volatility of financial series for clustering tasks.

Characterization of an H7N9 Influenza Virus Isolated from Camels in Inner Mongolia, China.

The H7N9 subtype of influenza virus can infect birds and humans, causing great losses in the poultry industry and threatening public health worldwide. However, H7N9 infection in other mammals has not been reported yet. In the present study, one H7N9 subtype influenza virus, A/camel/Inner Mongolia/XL/2020 (XL), was isolated from the nasal swabs of camels in Inner Mongolia, China, in 2020. Sequence analyses revealed that the hemagglutinin cleavage site of the XL virus was ELPKGR/GLF, which is a low-pathogenicity molecular characteristic. The XL virus had similar mammalian adaptations to human-originated H7N9 viruses, such as the polymerase basic protein 2 (PB2) Glu-to-Lys mutation at position 627 (E627K) mutation, but differed from avian-originated H7N9 viruses. The XL virus showed a higher SA-α2,6-Gal receptor-binding affinity and better mammalian cell replication than the avian H7N9 virus. Moreover, the XL virus had weak pathogenicity in chickens, with an intravenous pathogenicity index of 0.01, and intermediate virulence in mice, with a median lethal dose of 4.8. The XL virus replicated well and caused clear infiltration of inflammatory cells and increased inflammatory cytokines in the lungs of mice. Our data constitute the first evidence that the low-pathogenicity H7N9 influenza virus can infect camels and therefore poses a high risk to public health. IMPORTANCE H5 subtype avian influenza viruses can cause serious diseases in poultry and wild birds. On rare occasions, viruses can cause cross-species transmission to mammalian species, including humans, pigs, horses, canines, seals, and minks. The H7N9 subtype of the influenza virus can also infect both birds and humans. However, viral infection in other mammalian species has not been reported yet. In this study, we found that the H7N9 virus could infect camels. Notably, the H7N9 virus from camels had mammalian adaption molecular markers, including altered receptor-binding activity on the hemagglutinin protein and an E627K mutation on the polymerase basic protein 2 protein. Our findings indicated that the potential risk of camel-origin H7N9 virus to public health is of great concern.

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K.

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P â€‹+ â€‹M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P â€‹+ â€‹M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.

Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China.

H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010-2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605-4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.

Genetically Related Avian Influenza H7N9 Viruses Exhibit Different Pathogenicity in Mice.

Avian influenza viruses can cross species barriers and adapt to mammals. The H7N9 subtype AIV that emerged in China in 2013 caused 1568 human infections, with a mortality rate of nearly 40%. We conducted a retrospective analysis of H7N9 viruses that were isolated in live poultry markets in 2013. We found that two avian-origin H7N9 isolates, A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013, have a similar genetic background but exhibit different pathogenicity in mice. Whole-genome alignment of the two H7N9 viruses was carried out, and only six amino acid differences mapped in five genes, including the well-known virulence molecular marker PB2-E627K. Our retrospective analysis highlighted the importance of monitoring the adaptive mutations in avian influenza viruses with zoonotic potential.