A novel avian influenza A(H7N9) infection emerged to cause an outbreak in the Yangtze River delta in early 2013, subsequently spreading to other provinces in China [1-4]. In the first wave of influenza A(H7N9) infections from February to July 2013, 135 patients were reported from 11 provinces and municipalities in China, leading to 45 deaths [5]. Further cases have been reported since October 2013. Genomic analysis revealed that the novel H7 haemagglutinin is genetically distinct from other historical and contemporary human influenza viruses [3,6]. Adverse clinical outcomes have been associated with co-existing medical conditions or the development of drug resistance [7,8]. Previous H7 subtype influenza virus infections in humans such as the influenza A(H7N7) outbreak in Netherlands in 2003 were poorly immunogenic and serodiagnosis and seroepidemiology were challenging [9]. We explored the kinetics of the serological responses to this novel virus in haemagglutination inhibition (HI) assays and in a recently developed H7N9 pseudotype virus particle neutralisation (Nab) test [10]. Viral pseudotypes have been previously shown to provide reliable correlation with conventional microneutralisation tests for influenza A(H5N1) serological studies [11]. We investigated correlations between serological responses and clinical outcome.

Methods

Patients and samples
In April 2013, 18 patients confirmed with influenza A(H7N9) infection by real-time PCR were hospitalised at the Shanghai Public Health Clinical Center (SHAPHC). Serum specimens were collected every three to four days following admission with two to seven serial serum samples being collected from each patient. Clinical data including patient demographic information, treatment, clinical investigations and disease progression were retrieved from the clinical notes. Written informed consent was obtained from all participants. The overall study was reviewed and approved by the Ethics Committee of SHAPHC.

Haemagglutination inhibition assay
The methods used were as previously described and used horse erythrocytes [12]. Serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken Co Ltd., Tokyo, Japan) to remove non-specific inhibitors. Stored serum samples collected in 2009 from individuals not infected with influenza were used as negative serum controls. The virus strain used was A/Shanghai/4664T/2013 (H7N9) (GenBank accession No: KC853228.1).

Pseudovirus-based neutralisation assay
To rapidly and safely assess neutralisation activities against the 2013 influenza A(H7N9) virus which caused severe disease in humans, we developed a luciferase reporter-based Nab assay which has a non-replicative human immunodeficiency virus backbone carrying influenza A H7 and H9. We have previously demonstrated that the titres quantified by Nab assay correlated well with the titres measured by traditional HI assay, using serum samples from influenza A(H7N9)-infected patients and uninfected subjects with good correlation (Spearman r=0.88) [10]. The pseudoviruses were prepared as described in our previous report [10]. The neutralising titre of human sera was defined as the highest serum dilution that gave ≥80% inhibitory concentration (IC80) of the luciferase signal in virus-infected MDCK cells. On the basis of previous studies we had defined that IC80 and a antibody titre of 1:40 were the best discriminators between patients and non-infected controls, and we employed these to define positive Nab responses.

Statistical analyses
Non-parametric Mann–Whitney test was used to test the differences in HI or Nab titres across groups. Categorical variables were compared by using the two-tailed Fisher’s exact test to account for small sample size. In addition, univariate and multivariate exact logistic regression modelling were employed to identify the association of different factors with clinical outcome and allow for small cell size. The covariates used in the multivariate model included age, sex and NAb titres (1:40 and 1:640). The results were presented using odds ratios (ORs).

We fitted accelerated failure time model assuming a Gaussian distribution to compare time from illness onset to reaching a Nab titre of 1:40 between patients who recovered versus those who died, accounting for interval censoring due to time of testing. The model was also used to identify factors associated with longer time to recovery for recovered patients. We compared the initial Nab titre and rate of increase in Nab titre, adjusted for age and sex, using a linear mixed model to account for repeated measurements, assuming a linear increasing trend by days since illness onset. For analyses based on continuous measurements, titres were first log-transformed (with base 10). We used bootstrap method with 1,000 resamples to test the difference in time from illness onset to reaching a Nab titre of 1:40 between fatal cases and survivors. All statistical tests were considered significant at the level of p<0.05. All data were analysed by using SPSS software (version 17.0) and R (version 3.0.1).

Results

To understand the kinetics of the human serological responses to the novel influenza A(H7N9) virus, we first determined (HI) antibody and pseudotype Nab responses in 18 influenza A(H7N9)-infected patients. HI antibody titres reached a titre of 1:40 in six of 14 patients by Day 10 of illness. By Day 18 of illness, 17 of 18 patients had antibody titres of 1:40 with titres ranging from <1:10 to 1:320, and 10 of 18 patients had titres ≥1:80. All patients had evidence of seroconversion within three weeks (Figure). We next examined Nab titres quantified by the Nab assay. Nab titres reached 1:40 in five of 14 patients by Day 10 of illness and 16 of 17 by Day 18 of illness. Thirty-seven control sera collected in 2009 had negative results in both tests. To test the reliability of assays, limited repeat testing has been done on sets of sera by both assays with good reproducibility; in addition, all the sera were tested in one large assay batch to maintain comparability.