Syndromic Surveillance in Humans

Since April 16, 2013, enhanced surveillance for influenza A(H7N9) virus has been conducted in 28 sentinel hospitals and 23 collaborating laboratories in 21 cities in Guangdong Province, China. All specimens are first tested for avian influenza A virus; specimens with positive results are then further tested in the laboratories of the 21 city CDCs for subtypes H5, H7, and H9 by using commercial real-time reverse transcription PCR (RT-PCR) (Liferiver, Shanghai ZJ Bio-Tech, Shanghai, China). Results are further verified at the Guangdong Provincial CDC. H7-positive specimens are tested for N9 gene segments by using real-time RT-PCR (2). H7N9-positive swab materials and sputum samples are then blindly passaged for 2 or 3 generations in 9- or 10-day-old embryonated chicken eggs for virus isolation. Hemagglutination-positive allantoic fluids are collected, and viruses are subtyped by hemagglutination and neuraminidase inhibition (HI and NI) assays by using a panel of reference antiserum, as described by Huang et al. (10). Standard precautions are taken to avoid cross-contamination of specimens. All H7 influenza viruses isolated from patients undergo complete genome sequencing.

Environmental Surveillance

In April 2013, the Guangdong Provincial CDC launched an environmental surveillance program to monitor for avian influenza viruses in LPMs. Environmental samples were collected from LPMs in Guangdong Province during April 15, 2013–February 28, 2014. Each week during April 15–May 31, 2013, twenty environmental samples per market were collected from selected markets in 21 cities at the prefecture level. The environmental sampling was done by collecting wet-swab specimens from poultry feces, chicken epilator surfaces, chopping board surfaces, cage surfaces, and sewage. If a human H7N9 case was confirmed and the case-patient had exposure in an LPM, >20 environmental samples would be collected from that market.

Samples were collected from poultry feces by swabbing the surfaces of the chicken epilator, chopping boards, and cages 4–8 times with separate cotton-tipped swabs (Copan Italia, Brescia, Italy). The swabs were then inserted into a tube containing 3 mL of virus transport medium (Copan Italia) and stored at 4°C before shipping to the CDC laboratory. At arrival in the laboratory, samples were vortexed and swabs were discarded. Total RNA and DNA were extracted simultaneously from 200 μL supernatant of the sample by using the QIAamp minElute Virus Spin Kit (QIAGEN, Crawley, UK), according to the manufacturer’s instructions. Then, 5 μL of eluate was tested for influenza A; thereafter, testing for subtypes H5, H7, and H9 was done by commercial real-time reverse transcription PCR (RT-PCR) (Liferiver, Shanghai ZJ Bio-Tech). H7-positive specimens were further subjected to N9 gene-specific RT-PCR. Dual positive samples were blindly passaged for 2–3 generations in 9- to 10-day-old embryonated chicken eggs for virus isolation. Hemagglutinin (HA)–positive isolates were collected and further subtyped by HI and NI assays using a panel of reference antiserum, as described by Huang et al. (10). Standard precautions were taken to avoid cross-contamination of specimens. Monoinfluenza virus infection (without mixed infection of other subtypes) was further confirmed by RT-PCR before whole-genome sequencing. All H7N9 isolates from LPMs were subjected to complete genome sequencing, as were 7 H9 influenza viruses that were isolated from different LPMs during May–December 2013.

Whole-Genome Deep Sequencing

For all selected isolates, all 8 gene segments (HA, neuraminidase [NA], nucleoprotein [NP], polymerase basic 1 [PB1] and 2 [PB2], polymerase acidic [PA], matrix [M], and nonstructural [NS]) were sequenced by using a next-generation sequencing strategy for influenza A virus sequencing with the Ion PGM System and PathAmp FluA Reagents (Life Technologies, Carlsbad, CA, USA). Viral RNA extraction was performed by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription PCR amplification of all 8 gene segments were performed by using PathAmp Flu A Preamplification Reagents (Life Technologies). Amplicons were purified and quantitated by using the Ampure XP purification kit (Beckman, Brea, CA, USA). The genomic libraries were prepared with the Ion Xpress Plus Fragment Library Kit (Life Technologies). Enzymatic fragmentation was used for the 200-bp read protocol with a 10-min incubation time. Samples were assigned barcodes by using the Ion Xpress Barcode Adapters 1–32 Kit (Life Technologies). Automated template preparation was performed by using the Ion OneTouch 2 System (Ion PGM Template OT2 200 Kit; Life Technologies). Final sequencing was performed with the Ion PGM Sequencing 200 Kit v2 (Life Technologies) using the Ion 316 Chip V2.

Genome Sequence Alignment

Genome sequence assembly was done with the pathogen analysis program of the Ion PGM server (Life Technologies). Multiple sequence alignment against previously published complete genome sequences of H7N9 and H9N2 viruses was performed by using ClustalW (http://www.clustal.org) and BioEdit software version 7.14 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The whole-genome sequences were submitted to GISAID (http://www.gisaid.org; accession nos. EPI148417 and EPI151429–151437).

Phylogenetic Analysis and Data Visualization

Maximum-likelihood trees were estimated for all 8 gene segments by using MEGA version 5.2 (http://www.megasoftware.net) with the general time reversible + Γ model (11). To assess the robustness of individual nodes on phylogenetic trees, a bootstrap resampling process (1,000 replications) and the neighbor-joining method were used.

The initial trees were constructed in PhyML by using general time reversible + I + Γ4 as the evolutionary model (12). GARLI (https://code.google.com/p/garli/) was run on the best tree from PhyML for 1 million generations to optimize tree topology and branch lengths.

Syndromic Surveillance

A total of 34,342 samples from patients with pneumonia or influenza-like illness from 21 cities in Guangdong Province were tested during April 2013–February 2014. Of these, samples from 81 patients from 10 cities tested positive for influenza A(H7N9) virus. With the exception of 1 case confirmed in August 2013, all other cases were found starting in October 2013. In total, 6 cases were confirmed in 2013 and 75 in early 2014.

Patient characteristics are summarized in Table 1. Most patients were male, and the median age of all patients was 52 years. Of the 81 patients, 70 (84%) had severe illness, which likely reflected the design of the surveillance (hospitalized patients). Twenty-two (27%) patients died; 14 remained hospitalized as of May 2014, and 45 had recovered. Overall, 73 patients (90.1%) reported poultry contact (Table 1).

Environmental Surveillance

Figure 1

Figure 1. Distribution of influenza A H9 (A) and H7 (B) viruses, Guandong Province, China, 2013–2014. Shading indicates percentage of environmental swab specimens from live poultry markets in each region that were positive...

A total of 8,942 swab samples from LPMs were collected and screened in laboratories of city CDCs at the prefecture level and at Guangdong Provincial CDC (2). Overall, 425 samples (4.8%) from 13 cities tested positive for H7 viruses, and 1,050 (11.8%) from 19 cities were positive for H9 viruses. The prevalence of viral genes in regions with positive findings ranged from 1%–14% of swab samples for H7 viruses and 1.7%–27.5% for H9. The H9 detection rate remained ≈12% throughout the year, except during June, when only 4% of samples tested positive. H9 viruses were detected throughout Guangdong Province (19 of the 21 cities; Figure 1, panel A). In contrast, only 1 H7-virus positive specimen was detected during March–September 2013 (in Meizhou in April), but detections of H7 viruses increased beginning in October: from 1 site in October, 3 in November, 8 in January, and 7 in February. H7 viruses were detected in the Pearl River Regions of Guangdong Province (Figure 1, panel B). Human cases were found in 10 of the 13 cities where environmental samples were positive for H7, and in 0 of the 8 cities where no H7 viruses were found in environmental samples (Figure 1).

Sequence Analysis

Internal Genes

Figure 2

Figure 2. Timed phylogenies of internal gene segments of influenza A(H7N9) viruses detected in 16 patients from Guangdong Province, China, 2008–2014. A) Polymerase basic 2; B) polymerase basic 1; C) polymerase acidic; D)...

Whole-genome sequences were determined for the H7N9 virus isolates from 16 patients and 6 environmental samples and for the 9 H9 viruses collected during environmental surveillance. Phylogenetic trees were constructed for each internal gene segment against all currently available H7N9 and H9N2 virus sequences from the National Center for Biotechnology Information and GISAID. Phylogenetic analysis of the whole-genome sequences showed the clear separation of PB2, PB1, and NS gene sequences of the Guangdong patient isolates from those detected from patients from eastern China (Figure 2).

Gene sequences on the same branch were reviewed to identify region of origin; these sequences matched internal genes from H9N2 viruses detected in southern China (Technical Appendix). M genes of all viruses clustered with the genes detected in viruses from the main H7N9 virus cluster in 2013, whereas diversity was seen in the PA (main 2013 variant and 2 additional lineages) and NP (2 lineages) genes. Combined, the H7N9 virus isolates that were sequenced appeared to represent at least 4 different reassortants (Table 2).