Contribution of the <i>csgA</i> and <i>bcsA</i> genes to <i>Salmonella enterica</i> serovar Pullorum biofilm formation and virulence

<p><i>Salmonella</i> biofilm formation is important to environmental stress resistance and virulence. However, the roles of the <i>csgA</i> and <i>bcsA</i> genes, which affect curli protein and cellulose production, respectively, in <i>Salmonella enterica</i> serovar Pullorum, are unknown. Here we constructed deletions in the <i>csgA</i> and <i>bcsA</i> genes in <i>S. enterica</i> serovar Pullorum strain S6702 and evaluated several aspects of biofilm formation and virulence. Δ<i>csgA</i> showed decreased production of curli fimbriae, while Δ<i>bcsA</i> had reduced cellulose production. Both mutants had a reduced ability to form biofilms. Δ<i>csgA</i> was reduced in adhesion and invasion to HeLa cells and exhibited decreased intracellular proliferation in HD11 macrophages. Δ<i>bcsA</i> exhibited increased proliferation in HD11 cells and replicated better in chicken spleens, as compared to the wild-type strain. Δ<i>csgA</i> virulence was attenuated in assays involving oral challenge of one-day-old chickens.</p

Primers for the mutagenesis of the NA and NS genes of the H5N1 AIV SY strain.

<p>Ba-NA-1^a, the restriction endonucleases site for BsaI is underlined.</p><p>Bm-NS-1^b<b>,</b> the restriction endonucleases site for BsmBI is underlined.</p

Real-time PCR primers for detection of the expression levels of immune-related genes in the PBMCs of mallard ducks.

<p>Real-time PCR primers for detection of the expression levels of immune-related genes in the PBMCs of mallard ducks.</p

Growth kinetics of the viruses in Vero, MDCK, CEF, and DEF cells.

<p>The cells were infected with the wild-type strain and the four rescue viruses at an MOI of 0.01 TCID₅₀/cell, and the culture media were harvested at the indicated times after infection. The virus titers at each time point are presented as the mean ± SD of duplicate experiments.</p

Virus shedding of oropharyngeal and cloacal swabs of mallard ducks inoculated with 0.1⁶ EID₅₀ H5N1 viruses.

<p>The oropharyngeal and cloacal swabs were collected for virus isolation from each group at 3, 5, and 7 days postinfection.</p>A<p>dpi: days postinfection.</p>B<p>Two out of three ducks were dead on day 6 postinfection.</p

Figure 4. Replication kinetics of SY and the four rescue viruses in mallard ducks.

<p>The virus titers in the lungs, livers, hearts, spleens, kidneys, and brains of mallard ducks inoculated intranasally with 10⁶EID₅₀/0.1 ml of SY and the four rescue viruses were determined. Each horizontal bar represents the mean virus titer in log₁₀ EID₅₀/g of tissue. The horizontal line indicates the lower limit of detection. Different lowercase letters indicate significant differences among SY and the four viruses infected groups on the same day postinfection (<i>P</i><0.05). For example, on day 3 postinfection, the mean viral titers in the lungs of A+S+ (a) and A+S− (ab) groups were significantly lower than that of A−S+ (c) and A−S− (c) groups, and there were no significant differences between the A+S− (ab) and SY (bc) groups. There were also no significant differences among the A−S− (c), A−S− (c) and SY (bc) groups.</p

Real-time RT-PCR quantification of the expression of immune-related genes in mallard PBMCs and growth kinetics of the viruses in mallard PBMCs.

<p>Mallard PBMCs in a six-well plate were inoculated with SY and the four rescue viruses at an MOI of 1 TCID₅₀/cell. The total RNA was extracted from the PBMCs at 8 h postinfection, and equal amounts of RNA (1 µg) from each sample were used for RT-PCR. The gene expression was normalized to the expression level of the GAPDH gene and is presented as the fold increase relative to the results observed with mock-treated cells. The data represent the mean fold changes ± SD (A and B). Mallard PBMCs were also infected with these viruses at an MOI of 1 copy/cell, and the supernatant and cells were harvested at 4 h, 8 h, and 24 h post-infection. These samples were determined by quantitative real-time PCR (qRT-PCR) using the primers for the matrix gene. The numbers of the viruses are presented as the mean ± SD of duplicate experiments (C).</p

Frequency of H5N1 viruses with double deletions in the NA and NS proteins from 1996 to 2012^a.

a<p>All available sequences of both NA and NS1genes from H5N1 viruses deposited in Genbank were selected.</p>b<p>The numbers indicate the ratio of the viruses to the total H5N1 viruses or the sources-based isolates in the indicated year.</p>c<p>Other sources: The viruses from wild birds, mammals (including humans), and environmental samples.</p>d<p>Percentage of H5N1 viruses with A− and S− isolated in the indicated year.</p

Enzymatic properties of the NA protein of H5N1 viruses.

a<p>The results are presented as the mean ± SD from three independent determinations on duplicate samples using dilutions of the H5N1 viruses.</p>b<p>V_max ratio of the rescue viruses to the wild-type SY virus.</p

Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses

<div><p>Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.</p></div