The index patient (Case 2) was a 4-year-old girl from downtown Wenshan. She developed high fever (39 ℃) on June 11, 2017. Two days later, she was admitted to a local hospital with acute tonsillitis. The patient was diagnosed as an influenza-like illness case, and a throat swab sample was collected and sent to the influenza network laboratory of the local CDC for routine surveillance. Avian influenza H7N9 virus RNA positivity was confirmed by the local CDC on June 20 using real-time RT-PCR. The symptoms were mild and the case was discharged from the hospital on June 24. During hospitalization, she was treated with antibiotics and febrifuge drugs without antiviral treatment. The patient had visited a local LPM on June 1, ten days before the onset of symptoms. (Figure 2)
Since the confirmation of the index patient, 4 additional human H7N9 infections were continually detected in June and July. Case 1 had high fever (40 °C) on June 9 and developed pneumonia. The case was confirmed as H7N9 virus infection via routine influenza surveillance work on June 23. Due to reports of two human H7N9 infections, the local CDC declared an H7N9 outbreak in the city. Nucleic acid detection of H7N9 virus was required by local hospital clinicians when they encountered patients with pneumonia not responding to antibiotic treatment. Cases 3, 4 and 5 were actively found by hospital doctors and confirmed by sending a swab sample to the local CDC. All the additional 4 cases had been exposed to local LPM. (Figures 1, 2) Furthermore, an epidemiological investigation showed that the chickens sold at the city markets were mainly originally transported from the adjacent Guangxi Province. (Figure 3 B)
Medical tracing was performed on 78 identified close contacts. All the close contacts were followed up for seven consecutive days, and no flu-like symptoms were noted among these people. Throat swabs were collected from the close contacts, and all tested negative for the H7 subtype.
There are five LPMs in downtown Wenshan (Figure 1). Six hundred forty-three samples containing poultry feces, poultry cage surface swabs and market sewage samples were collected from all five of the LPMs from June 20 to July 6. Thirty-nine of the samples tested positive for the H7 subtype by real-time RT-PCR and covered all five of the markets. However, the positive chicken and environmental samples failed to yield a virus isolate. After the positive detection in the LPMs, control measures including closure and disinfection of LPMs were performed from June 21 through July 7. Two hundred five environmental samples were collected again from these markets from July 13 through 21. All tested negative for the H7 subtype by real-time RT-PCR.
Viral amino acid substitutions
Full viral genomes were determined in four of five cases (except Case 5). These sequences were analyzed to identify the critical amino acid (aa) residue changes that may indicate their receptor specificity, drug resistance and mammalian adaptations. The HA proteins of all four isolated viruses presented a basic aa (KGR↓G) at the cleavage site, suggesting low pathogenicity in poultry. Substitutions at V186 and L226 in the HAs were identified, indicating an increased binding ability to human α 2,6-linked sialic acid receptors. No neuraminidase inhibitor (oseltamivir) resistance markers were present in the NA genes. All strains contained N31 in the M2 proteins, indicating amantadine resistance. Substitutions appeared at V368 in the PB1 protein, which was associated with an increased transmission in ferrets. (Table 1).
Phylogenetic analyses revealed that the HA genes of the four isolates from Wenshan patients were clustered in the same clade and were genetically similar to those from the infected patients in Guangxi Province in 2017. Additionally, the HA genes of the patient isolates were closely related to that of the isolate from the chicken sample in Wenshan City. (Figure 3 A, Box 1, Figure S1) However, the HA genes of the isolates from the cities of Wenshan and Kunming belonged to different branches in the phylogenetic tree. (Figure 3 A, Box 2) The Kunming strains were imported from Jiangxi Province. In addition, the Wenshan strains were transmitted from Guangxi Province by imported chickens. (Figure 3 B)
Identity of nucleotide sequences in viruses from Wenshan and Guanxi patients
Because the HA genes of the Wenshan viral strains were similar to those of the two isolates (A/Guangxi/6/2017 and A/Guangxi/18899/2017) from Guangxi Province by the above phylogenetic analysis, all eight viral gene segments were compared between the two isolate sources to determine the identities. The results showed that the nucleotide identity was over 99% in external genes (HA and NA) and was from 96 to 99% in six internal genes. (Table 2) The results showed that these viruses share a high degree of nucleotide sequence similarity.