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

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

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

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

Virulence comparison of the ten 2009 pandemic A (H1N1) influenza viruses in BALB/c mice.

<p>Mice were anesthetized and inoculated intranasally with virus (n = 23 per group; 50 µl of 10⁶ TCID₅₀). Ten randomly selected mice were monitored daily for signs of disease and mortality, up to 14 d.p.i. for (A), (B), and (C) research, whereas ten of the remaining mice from each group were euthanized at 5 d.p.i. to obtain lung tissue biopsies for use in (D). The final three mice were also sacrificed at 5 d.p.i. and their lungs were collected for (E) detection. (A) Survival percentage of mice. (B) Mean survival days for each challenged group. (C) Body weight changes of infected mice. Mean body weight and SD were calculated as percentage of body weight and compared to those at 0 d.p.i. (D) Viral RNA loads in lung tissues at 5 d.p.i. Data are presented as mean viral loads per microgram ± SD. (E) Viral titers in lung tissues at 5 d.p.i. Data are presented as mean log₁₀ PFU/mg. * <i>P</i><0.05 compared to the values of other viruses (one-way ANOVA).</p

Seroprevalence of Antibodies to Highly Pathogenic Avian Influenza A (H5N1) Virus among Close Contacts Exposed to H5N1 Cases, China, 2005–2008

<div><p>To assess the extent of highly pathogenic avian influenza (HPAI) A (H5N1) virus transmission, we conducted sero-epidemiologic studies among close contacts exposed to H5N1 cases in mainland China during 2005–2008. Blood specimens were collected from 87 household members and 332 social contacts of 23 H5N1 index cases for HPAI H5N1 serological testing by modified horse red-blood-cell hemagglutinin inhibition and microneutralization assays. All participants were interviewed with a standardized questionnaire to collect information about the use of personal protective equipment, illness symptoms, exposure to an H5N1 case during the infectious period, and poultry exposures. Two (2.3%) household contacts tested positive for HPAI H5N1 virus antibody, and all social contacts tested negative. Both seropositive cases had prolonged, unprotected, close contact with a different H5N1 index case, including days of bed-care or sleeping together during the index case’s infectious period, and did not develop any illness. None of the 419 close contacts used appropriate personal protective equipment including 17% who reported providing bedside care or having physical contact with an H5N1 case for at least 12 hours. Our findings suggest that HPAI H5N1 viruses that circulated among poultry in mainland China from 2005–2008 were not easily transmitted to close contacts of H5N1 cases.</p></div

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

Pathological analysis of the lung tissues of challenged BALB/c mice.

<p>Ten mice from each group were euthanized at 5 d.p.i. to obtain lung tissue biopsies, and for each lung three 4-µm sections were stained with H&E for pathological investigations. (A) Representative sections of H&E stained lung tissues from 10⁶ TCID₅₀ intranasally challenged mice. (B) Percentage of lesion area in lung tissues.</p

Direct binding assay with synthetic sialylglycopolymers.

<p>(A) Affinity to synthetic 3′SL or 3′DI-SL. (B) Affinity to synthetic 6′SLN or 6′DI-SLN.</p

The sensitivity and specificity of HI and MN assay for detection antibody to H7N9 virus.

<p>The sensitivity and specificity of HI and MN assay for detection antibody to H7N9 virus.</p