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.

Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus.

BACKGROUND: Encephalomyocarditis virus (EMCV) can infect a variety of animal species and humans. Although the EMCV infection is known to induce autophagy to promote its replication in host cells, the viral proteins that are responsible for inducing autophagy are unknown. METHODS: The recombinant plasmids that were expressing the EMCV proteins were constructed to analyze the role of each protein in the induction of autophagy. Autophagy inductions by the EMCV proteins in BHK-21 cells were investigated by confocal microscopy, Western blotting and transmission electron microscopy. ER stress in BHK-21 cells was examined by detecting the marker molecules using western blotting and luciferase assays. RESULTS: This study presents the first demonstration that the nonstructural proteins 2C or 3D of EMCV were involved in inducing autophagy in BHK-21 cells that were expressing 2C or 3D, and we found that inhibiting Beclin1 expression influenced this autophagy induction process. Next, 2C and 3D were shown to be involved in inducing autophagy by activating the ER stress pathway. Finally, EMCV 2C or 3D were demonstrated to regulate the proteins associated with PERK and ATF6alpha pathway. CONCLUSIONS: Our findings indicate that 2C and 3D are involved in EMCV-induced autophagy by activating ER stress molecules and regulating the proteins expression associated with UPR pathway, helping to better understand the EMCV-induced autophagy process.

Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

Construction of a one-step multiplex real-time PCR assay for the detection of serogroups A, B, and E of Pasteurella multocida associated with bovine pasteurellosis.

Bovine pasteurellosis, caused by serogroups A, B, and E of Pasteurella multocida (Pm), is mainly manifested as bovine respiratory disease (BRD) and hemorrhagic septicemia (HS). The disease has caused a great economic loss for the cattle industry globally. Therefore, identifying the Pm serogroups is critical for optimal diagnosis and subsequent clinical treatment and even epidemiological studies. In this study, a one-step multiplex real-time PCR assay was established. Three pairs of specific primers were prepared to detect the highly conserved genomic regions of serogroups A (HyaD), B (bcbD), and E (ecbJ) of Pm, respectively. The results depicted that the method had no cross-reaction with other bovine pathogens (Mannheimia hemolytica, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Salmonella Dublin, Mycobacterium paratuberculosis, infectious bovine rhinotracheitis virus, and Mycoplasma bovis). The linear range (107 to 102 copies/µL) showed the R2 values for serogroups A, B, and E of Pm as 0.9975, 0.9964, and 0.996, respectively. The multiplex real-time PCR efficiency was 90.30%, 90.72%, and 90.57% for CartA, CartB, and CartE, respectively. The sensitivity result showed that the serogroups A, B, and E of Pm could be detected to be as low as 10 copies/µL. The repeatability result clarified that an intra-assay and an inter-assay coefficient of variation of serogroups A, B, and E of Pm was < 2%. For the clinical samples, the detection rate was higher than the OIE-recommended ordinary PCR. Overall, the established one-step multiplex real-time PCR assay may be a valuable tool for the rapid and early detection of the serogroups A, B, and E of Pm with high specificity and sensitivity.

Application of a novel phage LPCS28 for biological control of Cronobacter sakazakii in milk and reconstituted powdered infant formula.

Contamination of infant formula with Cronobacter sakazakii (C. sakazakii) can cause fatal infections in neonates. Phages have emerged as promising antibacterial agents for food safety, but their effectiveness may be limited by thermal processing. In this study, we isolated 27 C. sakazakii phages from environmental water samples and selected LPCS28 due to its broad lysis spectrum. The titer of LPCS28 will not be significantly affected by heating at a temperature of 60 °C for one hour. In both reconstituted powdered infant formula (RPIF) and liquid milk, the pre-added LPCS28, after the thermal processing at 63 °C for 30 min, significantly inhibited the post-contaminated C. sakazakii (103 CFU/mL) and eventually reduced the number of C. sakazakii to below the limit of detection (<10 CFU/mL) within 9 h at 37 °C and significantly delayed the increase of bacterial concentration in the samples at 23 °C. The phylogenetic analysis revealed that LPCS28 belonged to a new genus, we proposed as Nanhuvirus, under the family Straboviridae. These findings suggest that phage LPCS28 is a promising biological control agent for pathogenic C. sakazakii in the dairy industry.

Characterization of four novel H5N6 avian influenza viruses with the internal genes from H5N1 and H9N2 viruses and experimental challenge of chickens vaccinated with current commercially available H5 vaccines.

Since 2014, highly pathogenic avian influenza H5N6 viruses have been responsible for outbreaks in poultry. In this study, four H5N6 virus strains were isolated from faecal samples of sick white ducks and dead chickens in Shandong in 2019. These H5N6 viruses were triple-reassortant viruses that have not been previously characterized. Their HA genes were derived from the H5 viruses and were closely related to the vaccine strain Re-11. Their NA genes all fell into the N6-like lineage and the internal gene were derived from H5N1 and H9N2 viruses. They all showed high pathogenicity in mice and caused lethal infection with high rates of transmission in chickens. Moreover, the SPF chickens inoculated with the currently used H5 (Re-11 and Re-12 strains)/H7 (H7-Re-2 strain) trivalent inactivated vaccines in China were completely protected from these four H5N6 viruses. Our study indicated the necessity of continued surveillance for H5 influenza A viruses and the importance of timely update of vaccine strains in poultry industry.

Human dendritic cell targeting peptide can be targeted to porcine dendritic cells to improve antigen capture efficiency to stimulate stronger immune response.

Dendritic cells (DCs) play a key role in the natural recognition of pathogens and subsequent activation of adaptive immune responses due to their potent antigen-presenting ability. Dendritic cell-targeting peptide (DCpep) is strongly targeted to DCs, which often express antigens, to enhance the efficacy of vaccines. Our previous study showed that recombinant Lactobacillus expressing human DCpep could significantly induce stronger immune responses than recombinant Lactobacillus without DCpep, but the mechanism remains unclear. In this study, the mechanism by which DCpep enhances the immune response against recombinant Lactobacillus was explored. Fluorescence-labeled human DCpep was synthesized to evaluate the binding ability of human DCpep to porcine monocyte-derived dendritic cells (Mo-DCs) and DCs of the small intestine. The effects of Mo-DC function induced by recombinant Lactobacillus expressing human DCpep fused with the porcine epidemic diarrhea virus (PEDV) core neutralizing epitope (COE) antigen were also investigated. The results showed that human DCpep bind to porcine DCs, but not to porcine small intestinal epithelial cells. Human DCpep can also improve the capture efficiency of recombinant Lactobacillus by Mo-DCs, promote the maturation of dendritic cells, secrete more cytokines, and enhance the ability of porcine DCs to activate T-cell proliferation. Taken together, these results promote advanced understanding of the mechanism by which DCpep enhances immune responses. We found that some DCpeps are conserved between humans and pigs, which provides a theoretical basis for the development of a DC-targeted vaccine.

Eurasian Avian-like M1 Plays More Important Role than M2 in Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

Reassortant variant viruses generated between 2009 H1N1 pandemic influenza virus [A(H1N1)pdm09] and endemic swine influenza viruses posed a potential risk to humans. Surprisingly, genetic analysis showed that almost all of these variant viruses contained the M segment from A(H1N1)pdm09, which originated from Eurasian avian-like swine influenza viruses. Studies have shown that the A(H1N1)pdm09 M gene is critical for the transmissibility and pathogenicity of the variant viruses. However, the M gene encodes two proteins, M1 and M2, and which of those plays a more important role in virus pathogenicity remains unknown. In this study, the M1 and M2 genes of A(H1N1)pdm09 were replaced with those of endemic H3N2 swine influenza virus, respectively. The chimeric viruses were rescued and evaluated in vitro and in mice. Both M1 and M2 of H3N2 affected the virus replication in vitro. In mice, the introduction of H3N2 M1 attenuated the chimeric virus, where all the mice survived from the infection, compared with the wild type virus that caused 100 % mortality. However, the chimeric virus containing H3N2 M2 was still virulent to mice, and caused 16.6% mortality, as well as similar body weight loss to the wild type virus infected group. Compared with the wild type virus, the chimeric virus containing H3N2 M1 induced lower levels of inflammatory cytokines and higher levels of anti-inflammatory cytokines, whereas the chimeric virus containing H3N2 M2 induced substantial pro-inflammatory responses, but higher levels of anti-inflammatory cytokines. The study demonstrated that Eurasian avian-like M1 played a more important role than M2 in the pathogenicity of A(H1N1)pdm09 in mice.

[Development of a reference substance for live bacterial count of Streptococcicosis live vaccines].

This study attempts to develop a reference substance for the live bacteria count of Streptococcicosis live vaccines in order to evaluate the validity of live bacterial count in inspection and testing. We prepared a batch of live Streptococcus suis reference substance for live bacterial count, tested their physical property, purity, vacuum degree, remaining moisture, and determined their homogeneity, thermal stability and transportation stability. Moreover, we organized collaborative calibration to assign count values to the reference substance and determine the shelf life of the reference substance in 12 months. The results showed that the physical property, the purity, the remaining moisture and the vacuum degree of the reference substance were all in compliance with the requirements of the Chinese Veterinary Pharmacopoeia. The homogeneity test showed that the coefficient of variation of the count of the reference substance was less than 10%, indicating a good homogeneity. Transportation stability test showed that the reference substance remained active after 72 h transportation in summer and winter with the package of styrofoam boxes and ice packs. Thermal stability test showed that the reference substance could be stored for up to 3 months at -20 °C, or up to 21 days at 4 °C. According to the collaborative calibration, the reference vaccine was assigned a count value range of (8.5-12.1)×107 CFU/ampoule. The shelf life test showed that the reference substance was stable for 12 months when stored at -70 °C. The reference substance could provide a reference for the live bacterial count of Streptococcicosis live vaccines. Moreover, it could also be used as a reference to evaluate the quality of corresponding agar media.

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.

Variation in the expression of Hsp27, αB-crystallin mRNA and protein in heart and liver of pigs exposed to different transport times.

Twenty pigs were randomly divided into four groups of five pigs each (not transported - control, 1, 2 and 4h of transportation). A significant increase of ALT, AST and CK in the blood serum and acute parenchyma cell lesions were observed and those were characterized by acute degenerations in the heart and liver. Hsp27 expression levels increased significantly in the heart after 2h and in the liver after 4h of transportation, accompanying with the hsp27 mRNA increasing significantly in the heart and liver after 1h of transportation. αB-crystallin expression levels were fluctuant (not significantly) in the heart and liver during transporting, however, αB-crystallin mRNA increase notably in the heart after 1h and decrease significantly in the liver at 1 and 2h of transportation, respectively. In conclusion, the cellular damage to the heart and liver is highest after 1h of transportation, Hsp27 and αB-crystallin play dissimilar roles and show tissue-specific response in different tissues during transportation.