Hydrophobic Residues at the Intracellular Domain of the M2 Protein Play an Important Role in Budding and Membrane Integrity of Influenza Virus.

M2 protein of influenza virus plays an important role in virus budding, including membrane scission and vRNP packaging. Three hydrophobic amino acids (91F, 92V, and 94I) at the intracellular domain of the M2 protein constitute a hydrophobic motif, also known as the LC3-interacting region (LIR), whereas the role of this motif remains largely unclear. To explore the role of the 91-94 hydrophobic motif for influenza virus, all three hydrophobic amino acids were mutated to either hydrophilic S or hydrophobic A, resulting in two mutant viruses (WSN-M2/SSS and WSN-M2/AAA) in the background of WSN/H1N1. The results showed that the budding ability of the M2/SSS protein was inhibited and the bilayer membrane integrity of the WSN-M2/SSS virion was impaired based on transmission electron microscopy (TEM), which in turn abolished the resistance to trypsin treatment. Moreover, the mutant WSN-M2/SSS was dramatically attenuated in mice. In contrast, the AAA mutations did not have a significant effect on the budding of the M2 proteins or the bilayer membrane integrity of the viruses, and the mutant WSN-M2/AAA was still lethal to mice. In addition, although the 91-94 motif is an LIR, knocking out of the LC3 protein of A549 cells did not significantly affect the membrane integrity of the influenza viruses propagated on the LC3KO cells, which suggested that the 91-94 hydrophobic motif affected the viral membrane integrity and budding is independent of the LC3 protein. Overall, the hydrophobicity of the 91-94 motif is crucial for the budding of M2, bilayer membrane integrity, and pathogenicity of the influenza viruses. IMPORTANCE M2 plays a crucial role in the influenza virus life cycle. However, the function of the C-terminal intracellular domain of M2 protein remains largely unclear. In this study, we explored the function of the 91-94 hydrophobic motif of M2 protein. The results showed that the reduction of the hydrophobicity of the 91-94 motif significantly affected the budding ability of the M2 protein and impaired the bilayer membrane integrity of the mutant virus. The mouse study showed that the reduction of the hydrophobicity of the 91-94 motif significantly attenuated the mutant virus. All of the results indicated that the hydrophobicity of the 91-94 motif of the M2 protein plays an important role in budding, membrane integrity, and pathogenicity of influenza virus. Our study offers insights into the mechanism of influenza virus morphogenesis, particularly into the roles of the 91-94 hydrophobic motif of M2 in virion assembly and the pathogenicity of the influenza viruses.

N-Linked Glycosylation Plays an Important Role in Budding of Neuraminidase Protein and Virulence of Influenza Viruses.

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knockout significantly inhibited the budding of NA. Further studies showed that the NLG knockout caused attenuation of virus in vitro and in vivo Notably, the NLG at 219 position plays an important role in the budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, the unfolded protein response (UPR) was determined in NLG knockout NA overexpressed cells, which showed that the mutant NA was mainly located in the endoplasmic reticulum (ER), the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knockout NA was stacked in the ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding.IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. In particular, the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

Detection and complete genome characteristics of Posavirus 1 from pigs in China.

Porcine stool-associated RNA virus 1 (Posavirus 1) is a novel member of picornaviruses and first identified from fecal samples of 30-day-old pigs with diarrhea in USA in 2011. To evaluate the existence of Posavirus 1 in swine herds, 118 clinical samples from diarrheal pigs and 31 fecal swabs from healthy pigs were collected and detected by reverse transcription-polymerase chain reaction (RT-PCR) using Posavirus 1-specific primers. Only five fecal samples from diarrheal pigs on two swine farms were positive for Posavirus 1. The complete genome sequences [excluding poly (A) tail] of two representative isolates SDQD-25 and HBTS-11 are determined and consist of 9840 and 9819 nucleotides in length, and encode one putative polyprotein of 3070 and 2952 amino acids, respectively. They share 90.3% homology with each other and 81.3-95.4% homologies with American Posavirus 1 isolates or strains at the nucleotide sequence level. The phylogenetic analysis based on the entire genomes of reference picornavirus strains or isolates showed SDQD-25, HBTS-11 cluster together with American Posavirus 1 isolates or strains, yet are clearly distant from the other picornaviruses. The complete genome sequences of Chinese Posavirus 1 isolates will enrich the information of Posavirus 1 sequence database and further expedite posavirus research on the genetic diversity, epidemiology, and evolution in China.

326K at E Protein Is Critical for Mammalian Adaption of TMUV.

Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses to the poultry industry in China since 2010. However, the potential threat of TMUV to mammals has not been well studied. In this study, a TMUV HB strain isolated from diseased ducks showed high virulence in BALB/c mice inoculated intranasally compared with the reference duck TMUV strain. Further studies revealed that the olfactory epithelium is one pathway for the TMUV HB strain to invade the central nervous system of mice. Genetic analysis revealed that the TMUV HB virus contains two unique residues in E and NS3 proteins (326K and 519T) compared with duck TMUV reference strains. K326E substitution weakens the neuroinvasiveness and neurovirulence of TMUV HB in mice. Remarkably, the TMUV HB strain induced significantly higher levels of IL-1ß, IL-6, IL-8, and interferon (IFN)-α/ß than mutant virus with K326E substitution in the brain tissue of the infected mice, which suggested that TMUV HB caused more severe inflammation in the mouse brains. Moreover, application of IFN-ß to infected mouse brain exacerbated the disease, indicating that overstimulated IFN response in the brain is harmful to mice upon TMUV infection. Further studies showed that TMUV HB upregulated RIG-I and IRF7 more significantly than mutant virus containing the K326E mutation in mouse brain, which suggested that HB stimulated the IFN response through the RIG-I-IRF7 pathway. Our findings provide insights into the pathogenesis and potential risk of TMUV to mammals.

Extraction optimization and screening of antioxidant peptides from grass carp meat and synergistic-antagonistic effect.

Grass carp (Ctenopharyngodon idellus) is one of the three most cultivated freshwater fish around the world, but it is mainly consumed afresh, so only a small part of them are processed into salted fish or snack food. This research was performed to prepare and screen antioxidant peptides from grass carp muscle to promote its high-value utilization. The parameters of double-enzyme two-step hydrolysis were optimized, the peptides with the highest ABTS.+ scavenging ability were enriched and identified by Sephadex G-25 and LC-Q-Orbitrap-MS/MS. The synergistic-antagonistic effect among identified peptides was also investigated. The optimized conditions were hydrolyzed with protamex (10,000 U/g) at pH 8.0, 50°C for 3 h, followed by hydrolysis with alcalase (6,000 U/g) at pH 9.0, 50 °C for 2 h, and the protein-liquid ratio was 4%. The hydrolysates were further fractionated to obtain five fractions, in which fraction 3 (F3) exhibited the strongest ABTS.+ and O 2 · - scavenging ability with the IC50 values of 0.11 and 0.47 mg/ml, respectively. Twelve novel antioxidant peptides were identified, in which VAGW possessed the highest activity (139.77 µmol GSH/g). Significantly synergistic effects were observed on the two and three peptides' combination among VAGW, APPAMW, LFGY, FYYGK, and LLLYK, while the C-terminal tryptophan (Trp) played an important role in the synergism. This study found that grass carp muscle hydrolysates can be potential natural antioxidants in functional products. The synergistic effects among peptides may provide a perspective for the combined application of peptides.

Platelet Activating Factor Receptor Exaggerates Microglia-Mediated Microenvironment by IL10-STAT3 Signaling: A Novel Potential Biomarker and Target for Diagnosis and Treatment of Alzheimer's Disease.

Background: Early diagnosis and effective intervention are the keys to delaying the progression of Alzheimer's Disease (AD). Therefore, we aimed to identify new biomarkers for the early diagnosis of AD through bioinformatic analysis and elucidate the possible underlying mechanisms. Methods and Results: GSE1297, GSE63063, and GSE110226 datasets from the GEO database were used to screen the highly differentially expressed genes. We identified a potential biomarker, Platelet activating factor receptor (PTAFR), significantly upregulated in the brain tissue, peripheral blood, and cerebrospinal fluid of AD patients. Furthermore, PTAFR levels in the plasma and brain tissues of APP/PS1 mice were significantly elevated. Simultaneously, PTAFR could mediate the inflammatory responses to exaggerate the microenvironment, particularly mediated by the microglia through the IL10-STAT3 pathway. In addition, PTAFR was a putative target of anti-AD compounds, including EGCG, donepezil, curcumin, memantine, and Huperzine A. Conclusion: PTAFR was a potential biomarker for early AD diagnosis and treatment which correlated with the microglia-mediated microenvironment. It is an important putative target for the development of a novel strategy for clinical treatment and drug discovery for AD.

Extraction optimization and screening of angiotensin-converting enzyme inhibitory peptides from Channa striatus through bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking.

Channa striatus is high-protein food with many health functions. This study aimed to prepare, screen and identify the angiotensin-converting enzyme inhibition peptides (ACEIPs) from C. striatus hydrolysates by response surface methodology and bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking. The optimal conditions for preparing ACEIPs were hydrolysis temperature 55 °C, hydrolysis time 3 h, pH 9, solid-liquid ratio 1:20 g/mL, and enzyme addition 5%, the ACE inhibition and molecular weight distribution of obtained hydrolysate was 54.35% and 8770-160 Da, respectively. Seven novel ACEIPs were screened through the established high-throughput screening approach, among which, EYFR and LPGPGP showed the strongest ACE inhibition with the IC50 value of 179.2 and 186.3 µM, respectively (P > 0.05). Molecular docking revealed that three and ten hydrogen bonds were formed between ACE and LPGPGP and EYFR, respectively, S1 and S2 were the major active pockets, but the major driving forces varied.

RNA-Seq Analysis of Influenza A Virus-Induced Transcriptional Changes in Mice Lung and Its Possible Implications for the Virus Pathogenicity in Mice.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA), where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

Genetic, antigenic and pathogenic characterization of avian coronaviruses isolated from pheasants (Phasianus colchicus) in China.

Two viruses were isolated in 2017 from commercial pheasants with severe clinical signs and mortality in Shandong and Anhui provinces, China, respectively. We examined the pathogenic effects of the viruses in chicken embryos and the size and morphology of the virus particles, performed phylogenetic analysis based on the S1 gene and complete genomic sequences, and examined the antibody responses against infectious bronchitis virus (IBV). The results suggested that the viruses I0623/17 and I0710/17 were avian coronaviruses and were identified as pheasant coronaviruses (PhCoV), with greatest similarity to IBV. Further investigations of the antigenicity, complete genome organization, substitutions in multiple genes, and viral pathogenicity, replication, and shedding in chickens and pheasants showed obvious differences between PhCoV and IBV in terms of antigenicity, and viral pathogenicity, replication, and shedding in chickens and pheasants. The close genetic relationship, but obvious differences between PhCoVs and IBVs suggested the IBVs could be the ancestors of PhCoVs, and that PhCoVs isolated from different outbreaks may have evolved independently from IBVs circulating in the specific region by adaption in pheasants. This hypothesis was supported by analysis of the S1 gene fragments of the two PhCoVs isolated in the current study, as well as PhCoVs isolated in the UK and selected IBV strains. Such analyses indicated different evolution patterns and different tissue tropisms between PhCoVs isolated in different outbreaks. Further studies are needed to confirm this hypothesis by studying the complete genomic sequences of PhCoVs from different outbreaks and the pathogenicity of IBVs in pheasants to compare and clarify the relationships between PhCoVs and IBVs.

Mechanisms of isoquercitrin attenuates ovalbumin glycation: Investigation by spectroscopy, spectrometry and molecular docking.

This research firstly investigated the inhibitory effect of isoquercitrin (ISQ) on Ovalbumin (OVA) glycation. The mechanism was elucidated through the interaction between OVA and ISQ, and changes in glycation sites and degree of each site as deduced by spectroscopy, spectrometry and molecular docking. ISQ significantly inhibited OVA glycation by attenuating the conformational change induced by glycation. It quenched the fluorescence of Trp via static mechanism, and exposed Trp residues to a more hydrophobic surroundings. Formation of OVA-ISQ complex was a endothermic processing driven by hydrophobic interactions, van der Waals forces and hydrogen bonds. LC-Orbitrap-MS/MS revealed that ISQ altered the location of glycation and alleviated the glycation degree of most sites. Molecular docking results indicated that ISQ inserted into the hydrophobic pocket of OVA with six hydrogen bonds and one π-π stacking formed between ISQ and the amino acid residues of OVA, leading to the altered glycation activity of some sites.

Mitigation of isoquercitrin on ß-lactoglobulin glycation: Insight into the mechanisms by mass spectrometry and interaction analysis.

Formation of advanced glycation end products (AGEs) on foods imposes threats to human health after intaking. This research firstly evaluated the inhibition of isoquercitrin on ß-lactoglobulin (ß-Lg) glycation, the mechanisms were elucidated by fluorescence spectroscopy, Orbitrap MSn and molecular docking. Fluorescence spectra indicated that isoquercitrin effectively alleviated the formation of AGEs, it could stabilize the conformation structure of glycated ß-Lg (G-ß-Lg), change the micro-environment in the vicinity of chromophores. SDS-PAGE analysis revealed the suppressed cross-linking of G-ß-Lg induced by isoquercitrin. The number of glycation site detected on G-ß-Lg was reduced from ten to eight after the addition of isoquercitrin, and the relative glycation degree of substitution of per site (RGDSP) of most glycation sites were also greatly decreased. As indicated by intermolecular interaction, isoquercitrin quenched the fluorescence of ß-Lg via a static mechanism, and their combination is an endothermic processing mainly derived by hydrophobic interaction, hydrogen bonds, and van der Waals forces. Isoquercitrin interacted with ß-Lg to form an equimolar complex, and one hydrogen bond was formed between isoquercitrin and Lys69 (4.96 Å). Above results proved that isoquercitrin can be a promising anti-glycation agent used in food system to prevent the formation of harmful glycation products.

Novel genotype of infectious bronchitis virus isolated in China.

Recombination events are known to contribute to the emergence of novel infectious bronchitis virus (IBV) genotypes. In this study, we carried out detailed phylogenetic analysis and sequence comparisons based on 74 complete nucleotide sequences of the IBV S1 gene, including strain I0636/16 and 73 representative sequences from each genotype and lineage. The results showed that strain I0636/16 represented a novel genotype, designated as lineage 1 within genotype VII (GVII-1). Further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage 18 within genotype I (GI-18)-like virus with an as-yet-unidentified sequence, likely derived from another IBV strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. To the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/CH/LGX/111119. These results emphasize the importance of limiting exposure to novel IBVs that may serve as a source of genetic material for emerging viruses, as well as the importance of IBV surveillance in chicken flocks.

Genetic and biological characteristics of four novel recombinant avian infectious bronchitis viruses isolated in China.

Infectious bronchitis viruses (IBVs) of GI-13 (793/B) and GI-19 (QX/LX4) lineages have been frequently detected in China in recent years. Naturally recombinant IBVs originating from the GI-13 and GI-19 lineages have also been isolated from chicken flocks with respiratory and renal problems in China. Thorough genetic and biological investigations of these recombinant viruses have led to speculation regarding their origin, evolution, and control. In order to confirm the previous results and further extend our understanding about the characteristics of the four recombinant IBV strains we had previously identified (I0718/17, I0722/17, I0724/17, and I0737/17), we conducted phylogenetic analysis by comparing their complete S1 gene sequences with those of 71 reference strains of different genotypes and lineages. We identified a close relationship between the S1 sequences of the four strains and those of GI-13 strains. The results of complete genome sequence analysis confirmed the previously identified recombination events in the four IBV strains and revealed additional recombination events in different genomic regions of strains I0718/17 and I0724/17, suggesting that the two strains originated from multiple recombination events between 4/91-like and YX10-like viruses. We comparatively evaluated the antigenicity, pathogenicity, and affinity of the four recombinant viruses and their deduced parental strains in the trachea and kidneys. Some of the strains showed comparable antigenic relatedness, pathogenicity, and affinity for the trachea and kidneys among each other and with their parental viruses; however, some of them showed varying biological characteristics. Point mutations observed in the receptor-binding domain and hypervariable region of the S1 subunit of the spike protein likely have an effect on these differences in biological characteristics, although the influence of other factors-such as host innate-immune responses and changes in genomic regions beyond the S1 protein-might also be responsible for such changes.

Molecular and biological characteristics of the infectious bronchitis virus TC07-2/GVI-1 lineage isolated in China.

In the present study, a thorough comparison of the infectious bronchitis virus (IBV) TC07-2/GVI-1 linage was conducted by comparing the S1 gene sequences of GVI-1 viruses with those of viruses representing the established genotypes and lineages. IBV GVI-1 strains were found to be closely genetically related to each other, irrespective of where the viruses were isolated, and differed from other known IBV genotypes and lineages; thus, it was confirmed that GVI represents a novel genotype. However, the GVI-1 viruses exhibited variable antigenicity when compared to each other. Further analysis found that strains CO8089L and CO8091L, which were isolated in Colombia in 2003, were closely related to GVI-1 viruses, suggesting that GVI-1 viruses likely originated from Colombia and are prevalent in at least five countries (Colombia, China, the Republic of Korea, Japan, and Vietnam). Analysis of the complete GVI-1 virus genomes suggested that the GVI-1 strains in China may be independently derived from recombination events that occurred between GI-19 strains and CO8089L/CO8091L-like viruses following the introduction of the viruses from Colombia. Similar to the viruses isolated in the Republic of Korea, GVI-1 viruses isolated in China also showed an affinity for the respiratory tract of chickens, which differed from one of the deduced parental viruses, the GI-19 strain. This difference may be due to recombination events that occurred in the genomes of the GVI-1 viruses, resulting in the replacement of the spike gene sequences in an YX10-like strain of GI-19 lineage.