Emergence of waterfowl‐originated gene cassettes in HPAI H7N9 viruses caused severe human infection in Fujian, China

Abstract Background Highly pathogenic avian influenza (HPAI) A(H7N9) virus emerged and caused human infections during the 2016‐2017 epidemic wave of influenza A(H7N9) viruses in China. We report a human infection with HPAI H7N9 virus and six environmental isolates in Fujian Province, China. Methods Environmental surveillance was conducted in live poultry markets and poultry farms in Fujian, China. Clinical and epidemiologic data and samples were collected. Real‐time RT‐PCRs were conducted for each sample, and H7‐positive samples were isolated using embryonated chicken eggs. Full genomes of the isolates were obtained by next‐generation sequencing. Phylogenetic analysis and antigenic analysis were conducted. Results A 59‐year‐old man who raised about 1000 ducks was identified as HPAI H7N9 infection. Six HPAI H7 viruses were isolated from environmental samples, including five H7N9 viruses and one H7N6 virus. Phylogenetic results showed the human and environmental viruses are highly genetically diverse and containing significantly different gene constellation from that of other HPAI H7N9 previously reported. The internal genes derived from H7N9/H9N2, H5N6, and the Eurasian wild‐bird gene pool, indicating waterfowl‐originated genotypes, have emerged in HPAI H7N9/N6 viruses and caused human infection. Conclusion The new genotypes raise the concern that these HPAI H7 viruses might transmit back into migratory birds and spread to other countries as the HPAI H5Nx viruses. Considering their capability of causing severe infections in both human and poultry, the HPAI H7 viruses in this study pose a risk to public health and the poultry industry and highlight the importance of sustained surveillance of these viruses.

Since 2013, the three original reassortment low pathogenic avian influenza (LPAI) A(H7N9) viruses have emerged and caused five epidemic waves with over 1500 human cases in China. 1 In early 2017, human infections with a highly pathogenic avian influenza (HPAI) A(H7N9) virus were reported in Taiwan and Guangdong Provinces of China. 2,3 Phylogenic analyses showed that the HPAI H7N9 virus originated from the LPAI H7N9 virus. 4,5 The signature genetic difference between HPAI and LPAI H7N9 virus was an insertion of four amino acids at the cleavage site of the HA protein. 3,6 Biological assays both in vitro and in vivo indicated that this insertion caused a switch in the virulence of the H7N9 virus in poultry. [6][7][8] To date, most evidence supports the notion that the insertion event probably occurred in poultry in the Pearl River Delta region and that the Guangdong Province was the original location of the HPAI H7N9 virus. 4,5 The HPAI H7N9 virus might have emerged in mid-2016, according to a molecular clock model. 4

| Ethics statement
As a public health response to the outbreak, written informed consent of the study subjects could be waived according to Chinese law.

| Clinical and epidemiologic data collection
The clinical history and epidemiologic information were obtained from epidemiological investigation reports, conducted by local centers for disease control and prevention.

| Sample collection and isolation
Respiratory specimens from patients and environmental samples including swabs of cages, feces, chopping boards, sewage, and poultry drinking water from live poultry markets or farms were collected by local centers for disease control and prevention, according to the national influenza surveillance guidelines. Each sample was collected in individual vials, placed in transport medium with antibiotics, and packed on ice before it was sent to the laboratory for further processing. To isolate the virus, 9-to 11-day-old specific-pathogen-free embryonated chicken eggs were inoculated with H7-positive samples for 48-72 hours at 37°C in BSL-3 laboratory. The allantoic fluid was harvested. And the viral titer was determined by hemagglutination assay. The positive fluid was stored at −80°C.

| Phylogenetic analysis
The highly similar sequences detected with BLAST and the publicly

| The patient
The patient was a 59-year-old man with diabetes who raised about  After sequencing, one of them was identified as an LPAI H7N9 virus and six were identified as HPAI H7 viruses including five H7N9 viruses and one H7N6 virus (Table 1). These HPAI H7 viruses were from four surveillance sites and were all collected after August 2017 ( Figure 1 and Table 1).

| Molecular markers
As shown in Table 1

| Origins of eight segments of HPAI H7 viruses
To identify the origins of the genetic diversity, phylogenetic analyses were conducted for all eight segments with sequences from the GISAID EpiFlu™ database, which have a higher similarity with viruses in this study, by BLAST ( Figure 2). The HA genes of the human isolate and six environmental HPAI H7 isolates clustered with those of other HPAI H7N9 viruses ( Figure 2A)   China, spread to South Korea and Japan, and other continents by migratory birds. 21 The new genotype viruses still carried the HA 186V residue, which indicate its dual receptor binding property. And the mammalian-adapted mutation PB2 701N was found in the human isolates. In addition, they contained several HA1 mutations to the HPAI H7N9 candidate vaccine virus, which could affect the HA antigenicity. Since September 2017, H5/H7 bivalent vaccination was adopted as a major control strategy in poultry in Mainland China.

| D ISCUSS I ON
Antigenic drift of H7N9 virus could increase the possibility of H7 vaccine escapes in the near future, which is an additional risk for re-emergence of H7N9 virus.
Our findings suggest that the HPAI H7 virus could have derived its genetic diversity from extensive avian influenza viruses and an expanded host range, possessing a risk to public health and animal health. Although no human to human transmission was detected, considering their ability to cause severe illness in humans and domestic birds, the sustained close surveillance of HPAI H7 viruses with novel genotypes is essential. who provide the epidemiologic information and specimens.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.