Image_1_Deficiency of C-reactive protein or human C-reactive protein transgenic treatment aggravates influenza A infection in mice.jpeg

C-reactive protein (CRP) has been shown to be a potential candidate target in the immunotherapy of severe influenza A infection. However, it is unclear on the pathogenesis associated with CRP in influenza infections. Here, we used influenza A H1N1 CA04 to infect human CRP transgenic mice (KI), CRP knockout mice (KO), and wild-type mice (WT), respectively, and compared the viral pathogenicity and associated immune response in those mice. The results showed that CA04 infection resulted in 100%, 80%, and 60% death in KO, KI, and WT mice, respectively. Compared to WT mice, CA04 infection resulted in higher TCID50 in lungs on day 3 after infection but lowered HI antibody titers in sera of survivors on day 21 after infection in KI mice. ELISA assay showed that IFN-γ concentration was significantly increased in sera of WT, KI, or KO mice on day 7 after infection, and IL-17 was remarkably increased in sera of WT mice but decreased in sera of KI mice while no significant change in sera of KO mice on day 3 or 7 after infection. Quantitative RT-PCR showed that the relative expression levels of immune checkpoint CTLA-4, LAIR-1, GITR, BTLA, TIM-3, or PD-1 mRNA in the lung presented decreased levels on day 3 or 7 after infection in KI or KO mice. The correlation analysis showed that mRNA expression levels of the 6 molecules positively correlated with viral TICD50 in WT mice but negatively correlated with viral TCID50 in KI or KO mice. However, only LAIR-1 presented a significant correlation in each lung tissue of WT, KI, or KO mice with CA07 infection statistically. IHC results showed that LAIR-1 positive cells could be found in WT, KO, or KI mice lung tissues with CA04 infection, and the positive cells were mainly distributed in an inflammatory dense area. Our results suggested that deficiency of CRP or human CRP transgenic treatment aggravates influenza A virus infection in mice. CRP is a double sword in immune regulation of influenza infection in which IL-17 and immune checkpoint may be involved.</p

Database search for SC_PB2-H357N or PA-A36T mutation in virus isolates from nature.

<p>Polymorphisms of PA-36 and PB2-357 in influenza virus isolates were assessed and compared with our findings. The data shown are the number of sequences of different influenza subtypes in the NCBI database, the number of sequences of identical same amino acid composition as SC_WT and SC_M, and the number of sequences with a different amino acid at the same position (with the amino acids shown in parentheses). SC_M, SC_PB2-H357N/PA-A36T; Others*, subtypes which were not listed above; —, not applicable.</p

Growth properties of recombinant viruses in human (A, B), porcine (C, D) and murine cells (E, F).

<p>Confluent monolayer of A549, PK15 and LA-4 cell lines were inoculated with SC_WT, SC_PA-A36T or SC_PB2-H357N virus at MOI of 0.0001. Culture supernatants were harvested at 12, 24, 48, 60, 72 and 96 hpi at 35(<b>A, C, E</b>) or 39°C (<b>B, D, F</b>), respectively. Virus titers were determined by TCID₅₀ assay using MDCK cells. Results are presented as mean ± SEM and are representative of three determinations. *, °, <i>p</i><0.05, when comparing SC_PA-A36T and SC_PB2-H357N with SC_WT respectively, as determined by a <i>t</i>-test of TCID₅₀ values. **, °°, <i>p</i><0.001, as determined by <i>t</i>-test.</p

Viral RNA polymerase activity of SC_WT, SC_PA-A36T and SC_PB2-H357N in 293T cells cultured at different temperatures.

<p>Luciferase-based minigenome reporter assays were used to measure polymerase activity in 293T cells at 33, 37, or 39°C. Cells were co-transfected with Gluc reporter plasmid and expression plasmids PB1 and NP, PA, and PB2 (WT or PA-A36T, PB2-H357N mutants) to generate different viral RNPs. After culturing at 33, 37, or 39°C for 24 h, <i>Gaussia</i> luciferase production was measured. Results are presented as mean ± SEM and are representative of three determinations. **, <i>p</i><0.001, as determined by <i>t</i>-test.</p

Analysis of viral replication efficiency in the respiratory tracts of mice.

<p>Six-week-old female BALB/c mice (<i>n</i> = 3/group/time-point) were inoculated intranasally with 50 µl containing 10⁴ TCID₅₀ of SC_WT, SC_PA-A36T, and SC_PB2-H357N. Animals were euthanized at 12, 24, 48 and 72 hpi. The right lung of each animal was homogenized in PBS (1 ml) and then centrifuged. Viral titers in the supernatant from lung homogenates were determined by TCID₅₀ assay. Results are presented as mean ± SEM and are representative of three determinations. *, <i>p</i><0.05 and **, <i>p</i><0.001, as determined by <i>t</i>-test.</p

A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

<div><p>Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.</p></div

The information of test serum samples.

<p>Abbreviations: HI, hemagglutination inhibition assay; MN, microneutralization assay; WB, Western blot assay; GMT: geometric mean titers. WB*, eight serum samples were tested with the WB assay. WB**, one serum sample was used in the WB assay. Titers below 10 were considered negative and assigned a value of 5. 5 seasonal influenza viruses: H1N1, H3N2, H1N1 2009 pdm, B Victoria, B Yamagata.</p

Analysis of antibodies to H7N9 by age group.

<p>Abbreviations: HI, hemagglutination inhibition assay; MN, microneutralization assay; GMT: geometric mean titers.</p><p>Note: *, P<0.05 comparing with the GMT of the child group a and b values, respectively, according to <i>t</i>-tests.</p

Spectrum of antibodies against influenza A H7N9 virus by along the days after illness onset.

<p>Forty-seven serum samples were collected from 36 patients with H7N9 infection between April 2nd and June 28th were tested by both horse erythorocytes hemagglutinin inhibition (hRBC HI), the modified microneutralization (MN) to detect H7-specific antibody.</p

The comparation of sensitivity of standard MN and Modified MN, tRBC HI and hRBC HI with sera of H7N9 cases.

<p>Note: *, Using 20 sera with MN, HI titer and WB band (collected during 7–63 days).</p><p>95% CI was calculated using OpenEpi, Proportion using Mid-P Exact test.</p