Exploring the alternative virulence determinants PB2 S155N and PA S49Y/D347G that promote mammalian adaptation of the H9N2 avian influenza virus in mice.

The occurrence of human infections caused by avian H9N2 influenza viruses has raised concerns regarding the potential for human epidemics and pandemics. The molecular basis of viral adaptation to a new host needs to be further studied. Here, the bases of nucleotides 627 and 701 of PB2 were changed according to the uncoverable purine-to-pyrimidine transversion to block the development of PB2 627K and 701N mutations during serial passaging in mice. The purpose of this experiment was to identify key adaptive mutations in polymerase and NP genes that were obscured by the widely known host range determinants PB2 627K and 701N. Mouse-adapted H9N2 variants were obtained via twelve serial lung-to-lung passages. Sequence analysis showed that the mouse-adapted viruses acquired several mutations within the seven gene segments (PB2, PB1, PA, NP, HA, NA, and NS). One variant isolate with the highest polymerase activity possessed three substitutions, PB2 S155N, PA S49Y and D347G, which contributed to the highly virulent and mouse-adaptative phenotype. Further studies demonstrated that these three mutations resulted in increased polymerase activity, viral transcription and replication in mammalian cells, severe interstitial pneumonia, excessive inflammatory cellular infiltration and increased growth rates in mice. Our results suggest that the substitution of these three amino acid mutations may be an alternative strategy for H9N2 avian influenza viruses to adapt to mammalian hosts. The continued surveillance of zoonotic H9N2 influenza viruses should also include these mammalian adaptation markers as part of our pandemic preparedness efforts.

Development of an Enhanced High-Yield Influenza Vaccine Backbone in Embryonated Chicken Eggs.

Vaccination is an efficient approach to preventing influenza virus infections. Recently, we developed influenza A and B virus vaccine backbones that increased the yield of several vaccine viruses in Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cells. These vaccine backbones also increased viral replication in embryonated chicken eggs, which are the most frequently used platform for influenza vaccine manufacturing. In this study, to further increase the viral titers in embryonated chicken eggs, we introduced random mutations into the 'internal genes' (i.e., all influenza viral genes except those encoding the hemagglutinin and neuraminidase proteins) of the influenza A virus high-yield virus backbone we developed previously. The randomly mutated viruses were sequentially passaged in embryonated chicken eggs to select variants with increased replicative ability. We identified a candidate that conferred higher influenza virus growth than the high-yield parental virus backbone. Although the observed increases in virus growth may be considered small, they are highly relevant for vaccine manufacturers.

HNSPPI: a hybrid computational model combing network and sequence information for predicting protein-protein interaction.

Most life activities in organisms are regulated through protein complexes, which are mainly controlled via Protein-Protein Interactions (PPIs). Discovering new interactions between proteins and revealing their biological functions are of great significance for understanding the molecular mechanisms of biological processes and identifying the potential targets in drug discovery. Current experimental methods only capture stable protein interactions, which lead to limited coverage. In addition, expensive cost and time consuming are also the obvious shortcomings. In recent years, various computational methods have been successfully developed for predicting PPIs based only on protein homology, primary sequences of protein or gene ontology information. Computational efficiency and data complexity are still the main bottlenecks for the algorithm generalization. In this study, we proposed a novel computational framework, HNSPPI, to predict PPIs. As a hybrid supervised learning model, HNSPPI comprehensively characterizes the intrinsic relationship between two proteins by integrating amino acid sequence information and connection properties of PPI network. The experimental results show that HNSPPI works very well on six benchmark datasets. Moreover, the comparison analysis proved that our model significantly outperforms other five existing algorithms. Finally, we used the HNSPPI model to explore the SARS-CoV-2-Human interaction system and found several potential regulations. In summary, HNSPPI is a promising model for predicting new protein interactions from known PPI data.

Transforming acidic coiled-coil containing protein 3 suppresses influenza A virus replication by impeding viral endosomal trafficking and nuclear import.

Transforming acidic coiled-coil containing protein 3 (TACC3) is a motor spindle protein that plays an essential role in stabilization of the mitotic spindle. In this study, we show that the overexpression of TACC3 reduces the viral titers of multiple influenza A viruses (IAVs). In contrast, the downregulation of TACC3 increases IAVs propagation. Next, we map the target steps of TACC3 requirement to the early stages of viral replication. By confocal microscopy and nuclear plasma separation experiment, we reveal that overexpression of TACC3 results in a substantial decrease of IAV NP accumulation in the nuclei of infected cells. We further show that viral attachment and internalization are not affected by TACC3 overexpression and detect that the early and late endosomal trafficking of IAV in TACC3 overexpression cells is slower than negative control cells. These results suggest that TACC3 exerts an impaired effect on the endosomal trafficking and nuclear import of vRNP, thereby negatively regulating IAV replication. Moreover, the infection of different IAV subtypes decreases the expression level of TACC3 in turn. Consequently, we speculate that IAV ensures the generation of offspring virions by antagonizing the expression of inhibitory factor TACC3. Collectively, our results establish TACC3 as an important inhibitory factor for replication of the IAV, suggesting that TACC3 could be a potential target for the development of future antiviral compounds.

Comprehensive analysis of the key amino acid substitutions in the polymerase and NP of avian influenza virus that enhance polymerase activity and affect adaptation to mammalian hosts.

Accumulation of adaptive mutations in the polymerase and NP genes is crucial for the adaptation of avian influenza A viruses (IAV) to a new host. Here, we identified residues in the polymerase and NP proteins for which the percentages were substantially different between avian and human influenza viruses, to screen for key mammalian adaptive markers. The top 10 human virus-like residues in each gene segment were then selected for analysis of polymerase activity. Our research revealed that the PA-M311I and PA-A343S mutations increased the polymerase activity among the 40 individual mutations that augmented viral transcription and genomic replication, leading to increased virus yields, pro-inflammatory cytokine/chemokine levels and pathogenicity in mice. We also investigated the accumulative mutations in multiple polymerase genes and discovered that a combination of PB2-E120D/V227I, PB1-K52R/L212V/R486K/V709I, PA-R204K/M311I, and NP-E18D/R65K (hereafter referred to as the ten-sites joint mutations) has been identified to generate the highest polymerase activity, which can to some extent make up for the highest polymerase activity caused by the PB2-627 K mutation. When the ten-sites joint mutations co-occur with 627 K, the polymerase activity was further enhanced, potentially resulting in a virus with an improved phenotype that can infect a broader range of hosts, including mammals. This could lead to a greater public health concern than the current epidemic, highlighting that continuous surveillance of the variations of these sites is utmost important.

A Novel lncRNA SAAL Suppresses IAV Replication by Promoting Innate Responses.

Influenza A virus (IAV) infection has traditionally been a serious problem in animal husbandry and human public health security. Recently, many studies identified that long noncoding RNAs play an important role in the antiviral immune response after the infection of the influenza virus. However, there are still lots of IAV-related lncRNAs that have not been well-characterized. Using RNA sequencing analysis, we identified a lncRNA, named Serpina3i Activation Associated lncRNA (SAAL), which can be significantly upregulated in mice after IAV infection. In this study, we found that overexpression of SAAL inhibited the replication of A/WSN/33(WSN). SAAL upregulated Serpina3i with or without WSN infection. Overexpression of Serpina3i reduced influenza virus infection. Meanwhile, knockdown of Serpina3i enhanced the replication of WSN. Furthermore, knockdown of Serpina3i abolished the SAAL-mediated decrease in WSN infection. Overexpression of SAAL or Serpina3i positively regulated the transcription of interferon ß (IFN-ß) and several critical ISGs after WSN infection. In conclusion, we found that the novel lncRNA SAAL is a critical anti-influenza regulator by upregulating the mRNA level of Serpina3i.

TREX (transcription/export)-NP complex exerts a dual effect on regulating polymerase activity and replication of influenza A virus.

Influenza A viruses effectively hijack the intracellular "resources" to complete transcription and replication, which involve extensive interactions between the viral and host proteins. Herein, we screened the host factors, which belong to DExD/H-box protein family members, RNA-binding proteins or mitochondrial anchoring proteins, to investigate their effects on polymerase activity. We observed DDX39B and DDX39A, DEAD-box RNA-Helicases, exert a dual effect on regulating polymerase activity and replication of influenza A viruses. We further revealed that DDX39B and DDX39A interact with viral NP and NS1 proteins. Interestingly, the viral NP proteins could reverse the inhibitory effect of excess DDX39B or DDX39A on polymerase activity. Mechanistically, the TREX complex subunits, THOC1, THOC4 and CIP29, were recruited to DDX39B-DDX39A-NP complex in an ATP-dependent manner, via the interaction with DDX39B or DDX39A, followed by excess TREX-NP complexes interfere with the normal oligomerization state of NP depending on the ratio between the viral and host proteins. On the other hand, the TREX complex, an evolutionarily conserved protein complex, is responsible for the integration of several mRNA processing steps to export viral mRNA. Knockdown of TREX complex subunits significantly down-regulated viral titers and protein levels, accompanied by retention of viral mRNA in the nucleus. Taken together, screening the host factors that regulate the replication of influenza virus advances our understanding of viral pathogenesis and our findings point out a previously unclear mechanism of TREX complex function.

D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

An influenza virus vector candidate vaccine stably expressing SARS-CoV-2 receptor-binding domain produces high and long-lasting neutralizing antibodies in mice.

Viral infectious pathogens, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, can cause extremely high infection rates and mortality in humans. Therefore, it is urgent to develop an effective vaccine against coronavirus and influenza virus infection. Herein, we used the influenza virus as a vector to express the SARS-CoV-2 spike receptor-binding domain (RBD) and hemagglutinin-esterase-fusion (HEF) protein of the influenza C virus. We then evaluated the feasibility and effectiveness of this design strategy through experiments in vitro and in vivo. The results showed that the chimeric viruses could stably express the HEF protein and the SARS-CoV-2 spike RBD at a high level. BALB/c mice, infected with the chimeric virus, exhibited mild clinical symptoms, yet produced high specific antibody levels against RBD and HEF, including neutralizing antibodies. Importantly, high neutralizing antibodies could be retained in the sera of mice for at least 20 weeks. Altogether, our data provided a new strategy for developing safe and effective COVID-19 and influenza virus vaccines.

DDX5/METTL3-METTL14/YTHDF2 Axis Regulates Replication of Influenza A Virus.

DEAD-box helicase 5 (DDX5), a member of the DEAD/H-box helicases, is known to participate in all aspects of RNA metabolism. However, its regulatory effect in antiviral innate immunity during replication of influenza virus remains unclear. Herein, we found that human DDX5 promotes replication of influenza virus in A549 cells. Moreover, our results further revealed that DDX5 relies on its N terminus to interact with the nucleoprotein (NP) of influenza virus, which is independent of RNA. Of course, we also observed colocalization of DDX5 with NP in the context of transfection or infection. However, influenza virus infection had no significant effect on the protein expression and nucleocytoplasmic distribution of DDX5. Importantly, we found that DDX5 suppresses antiviral innate immunity induced by influenza virus infection. Mechanistically, DDX5 downregulated the mRNA levels of interferon beta (IFN-ß), interleukin 6 (IL-6), and DHX58 via the METTL3-METTL14/YTHDF2 axis. We revealed that DDX5 bound antiviral transcripts and regulated immune responses through YTHDF2-dependent mRNA decay. Taken together, our data demonstrate that the DDX5/METTL3-METTL14/YTHDF2 axis regulates the replication of influenza A virus. IMPORTANCE The replication and transcription of influenza virus depends on the participation of many host factors in cells. Exploring the relationship between viruses and host factors will help us fully understand the characteristics and pathogenic mechanisms of influenza viruses. In this study, we showed that DDX5 interacted with the NP of influenza virus. We demonstrated that DDX5 downregulated the expression of IFN-ß and IL-6 and the transcription of antiviral genes downstream from IFN-ß in influenza virus-infected A549 cells. Additionally, DDX5 downregulated the mRNA levels of antiviral transcripts via the METTL3-METTL14/YTHDF2 axis. Our findings provide a novel perspective to understand the mechanism by which DDX5 regulates antiviral immunity.

Virus-host interaction networks as new antiviral drug targets for IAV and SARS-CoV-2.

Currently, SARS-CoV-2, especially the Omicron strain, is ravaging the world and even co-infecting human beings with IAV, which is a serious threat to human public health. As of yet, no specific antiviral drug has been discovered for SARS-CoV-2. This requires deeper understandings of the molecular mechanisms of SARS-CoV-2-host interaction, to explore antiviral drug targets and provide theoretical basis for developing anti-SARS-CoV-2 drugs. This article discussed IAV, which has been comprehensively studied and is expected to provide the most important reference value for the SARS-CoV-2 study apart from members of the Coronaviridae family. We wish to establish a theoretical system for the studies on virus-host interaction. Previous studies have shown that host PRRs recognize RNAs of IAV or SARS-CoV-2 and then activate innate immune signaling pathways to induce the expression of host restriction factors, such as ISGs, to ultimately inhibit viral replication. Meanwhile, viruses have also evolved various regulatory mechanisms to antagonize host innate immunity at transcriptional, translational, post-translational modification, and epigenetic levels. Besides, viruses can hijack supportive host factors for their replication. Notably, the race between host antiviral innate immunity and viral antagonism of host innate immunity forms virus-host interaction networks. Additionally, the viral replication cycle is co-regulated by proteins, ncRNAs, sugars, lipids, hormones, and inorganic salts. Given this, we updated the mappings of antiviral drug targets based on virus-host interaction networks and proposed an innovative idea that virus-host interaction networks as new antiviral drug targets for IAV and SARS-CoV-2 from the perspectives of viral immunology and systems biology.

Antigenic Evolution Characteristics and Immunological Evaluation of H9N2 Avian Influenza Viruses from 1994-2019 in China.

The H9N2 subtype avian influenza viruses (AIVs) have been circulating in China for more than 20 years, attracting more and more attention due to the potential threat of them. At present, vaccination is a common prevention and control strategy in poultry farms, but as virus antigenicity evolves, the immune protection efficiency of vaccines has constantly been challenged. In this study, we downloaded the hemagglutinin (HA) protein sequences of the H9N2 subtype AIVs from 1994 to 2019 in China-with a total of 5138 sequences. The above sequences were analyzed in terms of time and space, and it was found that h9.4.2.5 was the most popular in various regions of China. Furthermore, the prevalence of H9N2 subtype AIVs in China around 2006 was different. The domestic epidemic branch was relatively diversified from 1994 to 2006. After 2006, the epidemic branch each year was h9.4.2.5. We compared the sequences around 2006 as a whole and screened out 15 different amino acid positions. Based on the HA protein of A/chicken/Guangxi/55/2005 (GX55), the abovementioned amino acid mutations were completed. According to the 12-plasmid reverse genetic system, the rescue of the mutant virus was completed using A/PuertoRico/8/1934 (H1N1) (PR8) as the backbone. The cross hemagglutination inhibition test showed that these mutant sites could transform the parental strain from the old to the new antigenic region. Animal experiments indicated that the mutant virus provided significant protection against the virus from the new antigenic region. This study revealed the antigenic evolution of H9N2 subtype AIVs in China. At the same time, it provided an experimental basis for the development of new vaccines.

The molecular determinants of antigenic drift in a novel avian influenza A (H9N2) variant virus.

BACKGROUND: In early 2020, a novel H9N2 AIV immune escape variant emerged in South China and rapidly spread throughout mainland China. The effectiveness of the current H9N2 vaccine is being challenged by emerging immune escape strains. Assessing key amino acid substitutions that contribute to antigenic drift and immune escape in the HA gene of circulating strains is critical for understanding virus evolution and in selecting more effective vaccine components. METHODS: In this study, a representative immune escape strain, A/chicken/Fujian/11/2020 (FJ/20), differed from current H9N2 vaccine strain, A/chicken/Anhui/LH99/2017 (AH/17) by 18 amino acids in the head domain in HA protein. To investigate the molecular determinants of antigenic drift of FJ/20, a panel of mutants were generated by reverse genetics including specific amino acids changes in the HA genes of FJ/20 and AH/17. The antigenic effect of the substitutions was evaluated by hemagglutination inhibition (HI) assay and antigenic cartography. RESULTS: Fujian-like H9N2 viruses had changed antigenicity significantly, having mutated into an antigenically distinct sub-clade. Relative to the titers of the vaccine virus AH/17, the escape strain FJ/20 saw a 16-fold reduction in HI titer against antiserum elicited by AH/17. Our results showed that seven residue substitutions (D127S, G135D, N145T, R146Q, D179T, R182T and T183N) near the HA receptor binding sites were critical for converting the antigenicity of both AH/17 and FJ/20. Especially, the combined mutations 127D, 135G, 145N, and 146R could be a major factor of antigenic drift in the current immune escape variant FJ/20. The avian influenza A (H9N2) variant virus need further ongoing epidemiological surveillance. CONCLUSIONS: In this study, we evaluated the relative contributions of different combinations of amino acid substitutions in the HA globular head domain of the immune escape strain FJ/20 and the vaccine strain AH/17. Our study provides more insights into the molecular mechanism of the antigenic drift of the H9N2 AIV immune escape strain. This work identified important markers for understanding H9N2 AIV evolution as well as for improving vaccine development and control strategies in poultry.

Genetic and biological properties of H10N3 avian influenza viruses: A potential pandemic candidate?

The continued emergence of human illness caused by avian influenza viruses (AIVs) demonstrates the threat of strains such as H5N1, H7N9, H10N8, and now H10N3. The genetic and biological properties of H10N3 viruses are not fully understood. In this study, three H10N3 strains isolated from live poultry markets (LPMs) were systematically studied. Genome sequencing showed that the poultry-origin viruses are highly homologous to the human H10N3 isolate. The three avian strains were A/chicken/Jiangsu/0146/2021(abbreviated as JS146, H10N3), A/chicken/Jiangsu/0169/2021 (JS169, H10N3), and A/chicken/Jiangsu/0189/2021(JS189, H10N3). Animal studies indicated that all three viruses are highly pathogenic to mice and that all could replicate efficiently in mouse nasal turbinate and lungs despite maintaining their avian receptor binding affinity. We also found that these viruses replicated efficiently in A549 cells and chicken embryos. The strain JS146 had sensitivity to the neuraminidase-targeting drugs oseltamivir and zanamivir, whereas JS169 and JS189 were more resistant; genetic comparison implied that a substitution at NA position 368 conferred drug resistance. Importantly, several key molecular markers associated with mammalian adaptation had been detected in both avian and human-isolated H10N3 influenza viruses in the HA (G228S), PB2 (I292V and A588V), PB1 (M317V and I368V), and PA (A343S, K356R and S409N) protein. The above work contributes new insight into the biology of this potentially zoonotic subtype and provides evidence supporting the continued epidemiological monitoring of human infections caused by AIV subtype H10N3.

SRSF3 facilitates replication of influenza A virus via binding and promoting the transport of viral mRNA.

Many host factors were involved in regulating the polymerase activity of influenza A virus. To fully explore the role of polymerase complex-related host factors, we combined high-throughput transcriptome data to analyze the changes in mRNA levels of these factors during viral infection. Transcriptome data showed that viral infection caused down-regulation of MYH9, HNRNPU, SRSF3 and RPS24 mRNA levels. We confirmed the changes in mRNA and protein levels of MYH9, HNRNPU and SRSF3 by qPCR and WB. Then their effects on virus replication were tested through overexpression and knockdown experiments. We emphatically explained the mechanism of SRSF3 during influenza virus replication. Results showed that SRSF3 promoted influenza virus replication and regulated viral protein expression at the post-transcriptional level. Further analysis found that SRSF3 regulated viral replication depends on the 88th amino acid. RIP and FISH experiments further proved that SRSF3 bound to viral mRNA and participated in the nuclear and cytoplasmic transport of viral mRNA. Collectively, these findings suggested that virus infection regulated the expression of many host factors and SRSF3 positively regulated virus replication.

Evolution of the PB1 gene of human influenza A (H3N2) viruses circulating between 1968 and 2019.

One avian H3N2 influenza virus, providing its PB1 and HA segments, reassorted with one human H2N2 virus and caused a pandemic outbreak in 1968, killing over 1 million people. After its introduction to humanity, the pandemic H3N2 virus continued adapting to humans and has resulted in epidemic outbreaks every influenza season. To understand the functional roles of the originally avian PB1 gene in the circulating strains of human H3N2 influenza viruses, we analyzed the evolution of the PB1 gene in all human H3N2 isolates from 1968 to 2019. We found several specific residues dramatically changed around 2002-2009 and remained stable through to 2019. Then, we verified the functions of these PB1 mutations in the genetic background of the early pandemic virus, A/Hong Kong/1/1968(HK/68), as well as a recent seasonal strain, A/Jiangsu/34/2016 (JS/16). The PB1 V709I or PB1 V113A/K586R/D619N/V709I induced higher polymerase activity of HK/68 in human cells. And the four mutations acted cooperatively that had an increased replication capacity in vitro and in vivo at an early stage of infection. In contrast, the backward mutant, A113V/R586K/N619D/I709V, reduced polymerase activity in human cells. The PB1 I709V decreased viral replication in vitro, but this mutant only showed less effect on mice infection experiment, which suggested influenza A virus evolved in human host was not always consisted with highly replication efficiency and pathogenicity in other mammalian host. Overall, our results demonstrated that the identified PB1 mutations contributed to the viral evolution of human influenza A (H3N2) viruses.

Mutations of 127, 183 and 212 residues on the HA globular head affect the antigenicity, replication and pathogenicity of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes devastating the poultry industry, has been circulating widely in the poultry population and causing huge economic losses. In this study, two H9N2 viruses with similar genetic backgrounds but different antigenicity were isolated from a poultry farm, namely A/chicken/Jiangsu/75/2018 (JS/75) and A/chicken/Jiangsu/76/2018 (JS/76). Sequence analysis revealed that their surface genes differed in three amino acid residues (127, 183 and 212) on the head of hemagglutinin (HA). To explore the differences between the two viruses in their biological features, six recombinant viruses, including the wild-type or mutant HA and NA of JS/75 and JS/76 were generated with A/Puerto Rico/8/1934 (PR8) backbone via reverse genetics. The chicken challenge study and HI assay data indicated that r-76/PR8 showed the most obvious antigen escape due to 127 and 183 amino acid substitutions in HA gene. Further studies verified that the 127N site was glycosylated in JS/76 and its mutants. Receptor-binding assays showed that all the recombination viruses were prone to bind the human-like receptors, except for the mutants which glycosylated 127N was deleted. Growth kinetics and mice challenge experiments indicated that 127N-glycosylated viruses showed less replication in A549 cells and lower pathogenicity in mice compared with wild-type viruses. Therefore, the glycosylation site and two amino acid alternations in the HA globular head were responsible for the differences in antigenicity and pathogenicity between the two H9N2 isolates. This study is significant in the research of the antigenic variation and vaccine updates for the H9N2 AIV. Also, highlighted the critical functions of glycosylation in the influenza virus on the pathogenicity against mammals.

Effect of rifaximin on gut-lung axis in mice infected with influenza A virus.

Gut-lung axis injury is a common finding in patients with respiratory diseases as well as in animal model of influenza virus infection. Influenza virus damages the intestinal microecology while affecting the lungs. Rifaximin, a non-absorbable derivative of rifamycin, is an effective antibiotic that acts by inhibiting bacterial RNA synthesis. This study aimed to determine whether rifaximin-perturbation of the intestinal microbiome leads to protective effects against influenza infection, via the gut-lung axis. Our results showed that influenza virus infection caused inflammation of and damage to the lungs. The expression of tight junction proteins in the lung and colon of H1N1 infected mice decreased significantly, attesting that the barrier structure of the lung and colon was damaged. Due to this perturbation in the gut-lung axis, the intestinal microbiota became imbalanced as Escherichia coli bacteria replicated opportunistically, causing intestinal injury. When influenza infection was treated with rifamixin, qPCR results from the gut showed significant increases in Lactobacillus and Bifidobacterium populations, while Escherichia coli populations markedly decreased. Furthermore, pathology sections and western blotting results illustrated that rifaximin treatment strengthened the physical barriers of the lung-gut axis through increased expression of tight junction protein in the colon and lungs. These results indicated that rifaximin ameliorated lung and intestine injury induced by influenza virus infection. The mechanisms identified were the regulation of gut flora balance and intestinal and lung permeability, which might be related to the regulation of the gut-lung axis. Rifaximin might be useful as a co-treatment drug for the prevention of influenza virus infection.

Analysis of the circRNAs expression profile in mouse lung with H7N9 influenza A virus infection.

Influenza A virus is a single-stranded RNA virus that can cause great mortality and economic loss worldwide. Circular RNAs (circRNAs) are non-coding RNAs that have been shown to have important functions in the regulation of biological processes. However, their functions during the influenza A virus infection process remain unclear. Herein, RNA sequencing technology was used to identify circRNAs expressed in mouse lungs during infection with H7N9/PB2-627 K/701D (H7N9/Wild-type) virus and PB2 mutant viruses (H7N9/PB2-627E/701D and H7N9/PB2-627E/701 N). We identified 7126 circRNAs at different genomic locations during H7N9 influenza virus and its mutant virus infections, of which 186 were differentially expressed. Enrichment analysis revealed that the differentially expressed circRNAs were associated with the viral infection process. Our study shows that circRNA expression profiles were altered following H7N9 influenza A virus infection and the differentially expressed circRNAs may have an important immune-regulating function during viral infection.

Hemagglutinin stalk-based monoclonal antibody elicits broadly reactivity against group 1 influenza A virus.

BACKGROUND: Influenza virus remains a continuous and severe threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. METHODS: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments. RESULTS: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What's more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy. CONCLUSIONS: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.