Genetic characterization of the first detected human case of avian influenza A (H5N6) in Anhui Province, East China

We compared complete genome sequences of two strains of an avian influenza A (H5N6) virus isolated from a patient in Anhui Province with those of other strains from GenBank and Global initiative on sharing all influenza data (GISAID). The HA gene of the isolated virus shared homology with that of A/chicken/Zhejiang/727155/2014 (H5N6) at the level of similarity of 98%. The six internal genes of the Anhui strains were close to those of H9N2 viruses from Zhejiang, Shandong, and Guangdong provinces, with a similarity of 99%. In addition, the similarity between the internal antigens (NP and MP) of the isolated H5N6 virus and H7N9 and H10N8 viruses was 99%. Based on the data of phylogenetic analysis, the H5N6 influenza virus isolated in Anhui Province belonged to clade 2.3.4.4. The virus was shown to have molecular characteristics of highly pathogenic avian influenza viruses, including eight glycosylation sites and an amino acid sequence of the HA protein cleavage site, PLRERRRKKR/GLF, containing multiple basic amino acids. Additionally, the stalk domain of the NA protein was found to have a deletion in NA stalk region (11 amino acids in N6, positions 58–68). Our study demonstrated that the H5N6 virus from Anhui Province represented a triple-reassortant virus and could be highly pathogenic to humans. The prevalence of this virus should be closely monitored.

Sequence analysis. All sequences were trimmed using the BioEdit 7.0 software, and a phylogenetic tree was constructed by using the neighbour-joining method of the Molecular Evolutionary Genetics Analysis software (MEGA 6.0). A dendrogram was obtained based on genetic distances. All reference sequences were downloaded from NCBI or GISAID.
Amino acid sequence analysis was performed using the BioEdit 7.0 software. To identify the ORF region, DNA sequences were translated into protein sequences. The translated amino acid sequences were aligned using the MEGA 6.0 software, and amino acid sites were analysed.

Results
Epidemiological characteristics. The patient was a 65-year-old female. Due to fever, chill, cough and other symptoms, she went to the Jianmin Hospital of Ningguo County on April 25, 2016. Clinical examination showed that the patient had a fever (38 °C), slightly swollen throat, shortness of breath in both lungs, and rightlung auscultation. WBC was 8.6 × 10 9 /L with 86% neutrophils. The patient was admitted to the hospital with unknown fever. The patient had a history of type II diabetes. She was treated with cefoxitin sodium and amikacin sulphate as anti-infection therapy, as well as with defervescence, phlegm-reducing and rehydration therapy for 2 days. Unfortunately, there was no obvious reduction of significant symptoms. On April 27, she was transferred to the Ningguo People's Hospital for further diagnosis and treatment. On April 28, she was suspected to be infected with an avian influenza virus after expert consultation at the Wuhu Yijishan Hospital in Anhui Province. At the same time, her blood samples were sent to the Anhui Provincial Center for Disease Control and Prevention. On April 29, the provincial CDC confirmed that the patient was infected with subtype H5N6 of avian influenza virus A, and her samples were sent to the Chinese National Influenza Center for further confirmation. According to an epidemiological investigation, the patient was exposed to a dead chicken and a normal chicken before the onset of the disease, and she processed and ate the dead chicken.
Analysis of similarity of gene fragments. We cultured the H5N6 virus from the upper and lower respiratory tract samples of the patient and isolated two strains of the virus (AH33162 and AH33163). Using Sanger sequencing on an ABI 3730XL analyser, we sequenced eight genomic fragments of each isolate and found 100% homology between the nucleotide sequences of the two strains. Strains with the highest sequence similarity, based on NCBI BLAST, are shown in The amino acid sequence of the HA protein cleavage site of the Anhui H5N6 influenza virus is PLRERRRKKR/ GLF, thus containing multiple basic amino acids and indicating that the virus possesses molecular characteristics of highly pathogenic avian influenza viruses. The stalk domain of the NA protein has a deletion in NA stalk region (11 amino acids in N6, positions 58-68). The H5N6 viruses from Guangzhou, Yunnan and Changsha have the same deletion at this position. However, the virus from Sichuan has no deletion, and the H274Y and R292K viral substitutions were not found, which suggests that the virus retains the sensitivity to neuraminidase inhibitors. However, the S31N substitution was detected in the M2 protein, suggesting the occurrence of drug resistance to amantadine. Additionally, E627K and L89V substitutions were found in the PB2 protein, normally seen in mammal-adapted AIVs, but no D701N substitution was detected. The I368V substitution occurred in the PB1 protein, associated with H5 transmissibility, but there was no H99Y substitution. We also found that N30D and T215A substitutions occurred in the M1 protein, and the P42S substitution occurred in the NS1 protein,    which were related to the virulence increase of the virus in mice, but no deletion of 80~84 amino acids was found (Table 3).

Discussion
Based on the genetic evolutionary relationships, the H5N6 influenza virus from Anhui Province represents a triple-reassortant virus. Its HA was derived from an H5 subtype avian influenza virus from clade 2.3.4.4, and NA was derived from subtype H5N6, H3N6 or H10N6. The six internal genes of the virus were derived from an H9N2 avian influenza virus. NAs of the H5N6 strains from Anhui, Guangzhou, Changsha and Yunnan, containing a deletion in NA stalk region (11 amino acids in N6, positions 58-68), are clearly in a different clade from that of NA of the Sichuan strain, with no deletion at the same sites, suggesting that there are two groups of H5N6 viruses, with different gene reassortment, which continue to evolve. It is difficult to speculate which H5N6 genotype will form the dominant strain.
In recent years, avian influenza viruses of subtype H5 from clade 2.3.4.4 have been very active. In addition to H5N6, there are subtypes H5N1, H5N2 and H5N8 forming a large gene pool, which facilitates the selection of different viral NAs for gene reassortment. During 2014~2015, there were fourteen reported cases of infection by these avian influenza viruses, which causes a widespread concern 9 . H5N6 viruses from clade 2.3.4.4 show a broad geographical distribution. Not only have they been found in many provinces in China, but the viruses were also isolated in neighbouring countries such as Laos, Vietnam and Japan. Recent studies have found that H5N6 viruses can infect wild birds and domestic cats 10 , suggesting that the bird migration may be an important factor causing the widespread prevalence of subtype H5, while the detection of infection of domestic cats implies that the virus may adapt to mammalian hosts upon effective multiplication. H9N2 viruses have provided six internal genes for H5N1, H7N9, and H10N8 viruses 11 . The six internal genes of the H5N6 virus isolated from the case in Anhui Province are highly homologous to those of subtype H9N2. Because of the current widespread occurrence of low pathogenic avian influenza viruses (H9N2), they are likely to form new virus subtypes with highly pathogenic avian influenza viruses, and there is a risk of an influenza pandemic.
Characterization of viral genes revealed six mutations that occurred in the receptor-binding site of the HA protein of the H5N6 virus from Anhui Province, in contrast with that of the H5N1 virus from clade 2.3.4; however, we did not find deglycosylation in our isolated strains, which enhances mammalian receptor affinity, at the 158 site 12 . In addition, we found no Q226L substitution, which is associated with mammalian receptor affinity 13 . The stalk domain deletion in the NA protein is associated with the enhancement of the viral replication ability and pathogenicity to mice, as well as with a better ability to be transmitted in poultry 14,15 . The PB2 protein with the E627K and L89V substitutions and PB1 with the I368V substitution are associated with increased adaptation to mammalian transmission and with viral replication 16,17 . The M1 protein with the N30D and T215A substitutions and NS1 with the P42S substitution can increase the viral virulence in mice 18,19 . We speculated that the isolated virus only acquired a partial ability to adapt to mammals but had molecular characteristics allowing it to widely spread in poultry. Meanwhile, the virus has many enhanced pathogenic mutations, and thus, its prevalence and pathogenic characteristics in animals should be closely monitored.
Outbreaks of human infections caused by new avian influenza viruses have continued in recent years, and for some viruses (H5N1 and H7N9), limited human-to-human transmission has been observed 20,21 . So far, there have been no reports of human-to-human transmission for subtype H5N6 of avian influenza viruses. It should be noted that this virus has been able to spread in live poultry, which plays an important role in the process of evolution of avian influenza viruses. The virus is likely to undergo further geographic spread in the future. Therefore, controlling the market of wild birds and live poultry and analysing virus evolution and gene mutations in a timely manner are essential.