Additional file 1: of Characteristics of influenza H13N8 subtype virus firstly isolated from Qinghai Lake Region, China

Phylogenetic trees of a (PB2), b (PA), c (NP), d (M), and e (NS) of two H13N8 viruses. Full sequences were used to conduct phylogenetic tree using MEGA 7 with 1000 neighbor-joining replicates. Two H13N8 viruses isolated in Qinghai lake region are indicated by filled circles. (PDF 1 MB

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

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 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

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

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

Serological survey of human serum samples for the presence of antibodies against AIV H5N1.

<p>Serological survey of human serum samples for the presence of antibodies against AIV H5N1.</p

Investigation of Avian Influenza Infections in Wild Birds, Poultry and Humans in Eastern Dongting Lake, China

<div><p>We investigated avian influenza infections in wild birds, poultry, and humans at Eastern Dongting Lake, China. We analyzed 6,621 environmental samples, including fresh fecal and water samples, from wild birds and domestic ducks that were collected from the Eastern Dongting Lake area from November 2011 to April 2012. We also conducted two cross-sectional serological studies in November 2011 and April 2012, with 1,050 serum samples collected from people exposed to wild birds and/or domestic ducks. Environmental samples were tested for the presence of avian influenza virus (AIV) using quantitative PCR assays and virus isolation techniques. Hemagglutination inhibition assays were used to detect antibodies against AIV H5N1, and microneutralization assays were used to confirm these results. Among the environmental samples from wild birds and domestic ducks, AIV prevalence was 5.19 and 5.32%, respectively. We isolated 39 and 5 AIVs from the fecal samples of wild birds and domestic ducks, respectively. Our analysis indicated 12 subtypes of AIV were present, suggesting that wild birds in the Eastern Dongting Lake area carried a diverse array of AIVs with low pathogenicity. We were unable to detect any antibodies against AIV H5N1 in humans, suggesting that human infection with H5N1 was rare in this region.</p></div

Sample collection sites around the Eastern Dongting Lake area.

<p>(A) Hunan Province. Dongting Lake is located to the north of Hunan Province (black circle). (B) Dongting Lake. The black circles indicate sample collection areas. Junshan district (green). Yueyang county (yellow) surrounding the Eastern Dongting Lake area. Villages where environmental samples from duck farms are shown in red.</p