Acetylation at K108 of the NS1 protein is important for the replication and virulence of influenza virus.

Non-structural protein 1 (NS1) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. In this study, an acetylation modification was identified at the K108 residue of the NS1 protein of H1N1 influenza virus. To further explore the function of the K108 acetylation modification of the NS1 protein, a deacetylation-mimic mutation (K108R) and a constant acetylation-mimic mutation (K108Q) were introduced into the NS1 protein in the background of A/WSN/1933 H1N1 (WSN), resulting in two mutant viruses (WSN-NS1-108R and WSN-NS1-108Q). In vitro and mouse studies showed that the deacetylation-mimic mutation K108R in the NS1 protein attenuated the replication and virulence of WSN-NS1-108R, while the constant acetylation-mimic mutant virus WSN-NS1-108Q showed similar replication and pathogenicity as the wild-type WSN virus (WSN-wt). The results indicated that acetylation at K108 of the NS1 protein has an important role in the replication and virulence of influenza virus. To further explore the potential mechanism, the type I interferon (IFN-I) antagonistic activity of the three NS1 proteins (NS1-108Q, NS1-108R, and NS1-wt) was compared in cells, which showed that the K108R mutation significantly attenuated the IFN-ß antagonistic activity of the NS1 protein compared with NS1-wt and NS1-108Q. Both NS1-wt and NS1-108Q inhibited the IFN-ß response activated by RIG-I CARD domain, MAVS, TBK1, and IRF3 more efficiently than the NS1-108R protein in cells. Taken together, the results indicated that acetylation at NS1 K108 is important for the IFN antagonistic activity of the NS1 protein and virulence of the influenza virus.

[Effect of Stx2-encoding phage on the motility and gene expression involved in moving of Escherichia coli lysogen].

OBJECTIVE: The effect of flhDC, fliA, fliD and fliE genes involved in moving of Escherichia coli (E. coli) on the motility of lysogened strain by Stx2-encoding phage phiMin27 was explored by gene knockout and phage lysogenic conversion. METHODS: Using the lambda Red recombinase system, the mutant strains of E. coli MG1655 named MG1655 deltaflhDC, MG1655 deltafliA, MG1655 deltafliD and MG1655 deltafliE were constructed. Then the corresponding complemented strains by ligating amplified targeted genes into the low copy vector pUC18 at the BamHI and Hind III sites and transforming these plasmids into mutant strains were acquired. By lysogenic infection of Stx2-encoding phage phiMin27, the lysogens for mutants named MG1655 deltaflhDCphiMin27, MG1655 deltafliAdeltaMin27, MG1655 deltafliDphiMin27 and MG1655 deltafliEphiMin27 were achieved. Subsequently, the motility of wild strain, the mutants, the complemented strains and the lysogens were detected. The changes of expression of the other genes involved in motility between wild strain and the lysogens before and after flhDC deletion by qRT-PCR were analyzed. RESULTS: Lysogenic infection of Stx2-encoding phage phiMin27 could promote the expression of fliA and fliD gene and enhance the motility of MG1655. For flhDC deletion, higher expression of fliA and fliD gene of MG1655 appeared, but the motility had no change. However, lysogen for MG1655 deltaflhDC lost the swimming motility. By gene transcriptional level detection, the expression of fliA and fliD gene of MG1655 deltaflhDCphiMin27 was down-regulated significantly compared with MG1655 deltaflhDC, and no marked variation was observed for fliE gene. The single deletion of fliA, fliD and fliE gene had no effect on the motility of E. coli MG1655 and lysogened strain by Stx2-encoding phage phiMin27. CONCLUSION: The results show that fliA and fliD gene together participated the regulation for flagella motility and flhDC gene could affect the motility of the lysogened strain by phage. It provides the theoretical basis for further research on the mutual regulation between phage lysogenization and host genes.

The Mobility of Eurasian Avian-like M2 Is Determined by Residue E79 Which Is Essential for Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.

Pre-exposure to Streptococcus suis improved survival of influenza virus co-infection in mice.

The synergism of the influenza virus and respiratory tract pathogens is known to exacerbate diseases in both humans and animals. The mechanism of the co-infection of associated respiratory tract pathogens is explored in this study. Co-infection has a directional effect when influenza virus or other pathogens occur in a different order. In the present study, we used a mouse animal model to study the synergism of influenza virus and Streptococcus suis co-infection in different orders of administration. We found that the group infected with bacteria alone did not show any clinical symptoms, but the group infected with the virus alone showed 100 % mortality and clinical signs typical in infected mice. In the bacteria infected following virus pre-exposure group, the mice died before the virus-infected group and showed severer clinical signs. When the influenza virus was administered after the bacteria, the infected mice showed reduced mortality compared with mice administered the influenza virus alone. The results indicated that the order of infection significantly affected the outcome of the co-infection of these two pathogens in the mice. However, the underlying mechanism was unclear. Therefore, a transcriptome analysis of mouse lungs was conducted to explore the potential mechanism. The results showed that inflammation and cell damage signaling pathways were upregulated, which may have contributed to the increased mortality in the secondary bacterial infection group. Upregulated innate immunity may have been a major cause of reduced mortality when the bacteria were inoculated before the virus infection.

Clustered, regularly interspaced short palindromic repeat (CRISPR) diversity and virulence factor distribution in avian Escherichia coli.

In order to investigate the diverse characteristics of clustered, regularly interspaced short palindromic repeat (CRISPR) arrays and the distribution of virulence factor genes in avian Escherichia coli, 80 E. coli isolates obtained from chickens with avian pathogenic E. coli (APEC) or avian fecal commensal E. coli (AFEC) were identified. Using the multiplex polymerase chain reaction (PCR), five genes were subjected to phylogenetic typing and examined for CRISPR arrays to study genetic relatedness among the strains. The strains were further analyzed for CRISPR loci and virulence factor genes to determine a possible association between their CRISPR elements and their potential virulence. The strains were divided into five phylogenetic groups: A, B1, B2, D and E. It was confirmed that two types of CRISPR arrays, CRISPR1 and CRISPR2, which contain up to 246 distinct spacers, were amplified in most of the strains. Further classification of the isolates was achieved by sorting them into nine CRISPR clusters based on their spacer profiles, which indicates a candidate typing method for E. coli. Several significant differences in invasion-associated gene distribution were found between the APEC isolates and the AFEC isolates. Our results identified the distribution of 11 virulence genes and CRISPR diversity in 80 strains. It was demonstrated that, with the exception of iucD and aslA, there was no sharp demarcation in the gene distribution between the pathogenic (APEC) and commensal (AFEC) strains, while the total number of indicated CRISPR spacers may have a positive correlation with the potential pathogenicity of the E. coli isolates.

Screening of single-stranded DNA (ssDNA) aptamers against a zearalenone monoclonal antibody and development of a ssDNA-based enzyme-linked oligonucleotide assay for determination of zearalenone in corn.

A hypotoxic immunosorbent assay for the detection of zearalenone (ZEN) was developed, by identifying a single-stranded DNA (ssDNA) aptamer with high specificity and affinity for a ZEN monoclonal antibody (mAb-ZEN). ssDNA aptamers, which could mimic ZEN epitopes, were identified using the modified systematic evolution of ligands by an exponential enrichment (SELEX) technique. The purified mAb-ZEN was coated on microtiter plates as a target recognized by the random oligonucleotide ssDNA library. The binding affinity between the aptamers and mAb-ZEN during each round was measured by the biotin­streptavidin­horseradish peroxidase system. During 15 rounds of screening, an increasing binding affinity was observed. The enriched ssDNA library binding to mAb-ZEN with high affinity was cloned, sequenced, and analyzed. One aptamer (number 46), which displays the highest affinity and specificity for the mAb-ZEN, was used to establish an indirect competition enzyme-linked oligonucleotide assay (ELONA) to measure the ZEN concentration in corn. Under optimal conditions, the regression equation for quantification of ZEN was y = −0.0778x + 0.713 (R2 = 0.9981). The detection limit and IC50 were 0.01 and 0.2 ng/mL, respectively, with a working range of 0.03­2.5 ng/mL. The recovery rates of the spiked samples in the ELONA ranged from 95 to 105%. Aptamers, which can mimic many types of low-weight analytes in agricultural products, could serve as surrogates for the development of hypotoxic, environmentally friendly immunological detection methods.

Rapid simultaneous quantification of zearalenone and fumonisin B1 in corn and wheat by lateral flow dual immunoassay.

A lateral flow dual immunoassay (LFDIA) was developed for rapid quantitative detection of zearalenone (ZEN) and fumonisin B1 (FB1) in corn and wheat samples on a single test strip. Two test lines and the control line on the nitrocellulose membrane were coated with ZEN and FB1 conjugates and goat anti-mouse IgG, respectively. Colloidal gold nanoparticles were conjugated with monoclonal antibodies against ZEN or FB1. The intensity of the test lines was analyzed by a photometric strip reader to determine the concentrations of ZEN and FB1 based on the calibration curves of known concentrations versus intensity readings. Test parameters such as types of buffers, ratio of the two gold-labeled antibodies, and dilution of the sample extracts and the gold-labeled antibodies were optimized. The detection limit was 0.35 and 5.23 ng/mL for ZEN and FB1, respectively, and the corresponding detection ranges were 0.94-7.52 and 9.34-100.45 ng/mL, respectively. Spiked and natural samples were analyzed using both LFDIA and liquid chromatography-tandem mass spectrometry. The two methods had a good correlation (R(2) = 0.96). The dual quantitative LFDIA is sensitive, rapid, and easy-to-use for on-site testing of a large number of samples.

Novel chemiluminescence immunoassay for the determination of zearalenone in food samples using gold nanoparticles labeled with streptavidin-horseradish peroxidase.

A novel highly sensitive chemiluminescence immunoassay (CLIA) was developed to detect zearalenone in food samples by using both biotinylated zearalenone conjugates and gold (Au) nanoparticles labeled with streptavidin-horseradish peroxidase for signal amplification. Biotinylated zearalenone-ovalbumin conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase were synthesized separately. The concentrations of immunoreagents and the reaction times of these immunoreagents were optimized to improve the performances of analytical methods. For the CLIA based on biotinylated zearalenone conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase, the limit of detection was 0.008 ng/mL and the IC50 was 0.11 ng/mL. The linear working range was 0.02-0.51 ng/mL. The cross-reactivities with the zearalenone analogues (α-zearalanol, zearalanone, α-zearalenol, ß-zearalanol, and ß-zearalenol) were 32, 17, 12, 0.3, and 0.1%, respectively. The recovery rates in spiked food samples were 97-117%, and the intraday and interday relative standard deviations were both <10%. Parallel analysis of natural food samples showed a good correlation between this novel CLIA and liquid chromatography-tandem mass spectrometry. This method provides a rapid, accurate, and highly sensitive method to determine levels of zearalenone in food samples.

Simultaneous quantitative determination of multiple mycotoxins in cereal and feedstuff samples by a suspension array immunoassay.

Mycotoxins produced by different species of fungi may coexist in single cereal and feedstuff samples, which could become highly toxic for humans and animals. In order to quantify four mycotoxins (zearalenone, fumonisin B1, deoxynivalenol, and aflatoxin B1) in cereal and feedstuff samples simultaneously, a new suspension array immunoassay was developed. Antimycotoxin monoclonal antibodies were conjugated to the surface of different encoding microspheres (19#, 37#, 39#, and 49#), and mycotoxin-protein conjugates were then coupled with biotin. Using streptavidin-phycoerythrin as a signal reporter protein, this direct competition multiple suspension array immunoassay was optimized. The results showed that the detection limits for zearalenone, fumonisin B1, deoxynivalenol, and aflatoxin B1 were 0.51, 6.0, 4.3, and 0.56 ng/mL, respectively, with detection ranges of 0.73-6.8, 11.6-110.3, 8.6-108.1, and 1.1-14.1 ng/mL, respectively. For the detection of the spiked samples, the recovery rates were between 92.3% and 115.5%. This method also shows a good correlation coefficient (r = 0.99, P < 0.01) with liquid chromatography-tandem mass spectrometry in the detection of toxins in commercial cereal and feedstuff samples. This suspension array immunoassay was high-throughput and accurate for the rapid quantitative detection of multiple mycotoxins in commercial cereal and feedstuff samples.

cvhA gene of Streptomyces hygroscopicus 10-22 encodes a negative regulator for mycelia development.

A five-gene cluster cvhABCDE was identified from Streptomyces hygroscopicus 10-22. As the first gene of this cluster, cvhA encoded a putative sensor histidine kinase with a predicted sensor domain consisting of two trans-membrane segments at the N-terminus and a conserved HATPase_c domain at the C-terminus. The C-terminus polypeptide of CvhA expressed in Escherichia coli was purified and shown to be autophosphorylated with [gamma-32P]ATP in vitro. The phosphoryl group was acid-labile and basic-stable, which supported histidine as the phosphorylation residue. No obvious difference of mycelia development was observed between the null mutant of cvhA generated by targeted gene replacement and the wild-type parental strain 10-22 grown on solid soya flour medium with 2%-8% glucose or sucrose, but the cvhA mutant could form much more abundant aerial mycelia and spores than the wild-type strain on solid soya flour medium supplemented with 6%-8% mannitol, 6%-8% sorbitol, 4%-6% mannose, or 4%-6% fructose. This phenotype was complemented by the cloned wild-type cvhA gene, and no difference was observed for growth curves of the cvhA mutant and the wild strain in liquid minimal medium with the tested sugars at a concentration of 4%, 6% and 8%. We thus propose that CvhA is likely a sensor histidine kinase and negatively regulates the morphological differentiation in a sugar-dependent manner in S. hygroscopicus 10-22.

Identification and characterization of hmr19 gene encoding a multidrug resistance efflux protein from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22.

The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane alpha-helical spanners, belonging to the drug:H(+) antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [(3)H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.