key: cord-0989208-u5092r08 authors: Bi, Fuyin; Jiang, Lili; Huang, Lihua; Wei, Jingguang; Pan, Xiaowen; Ju, Yu; Mo, Jianjun; Chen, Minmei; Kang, Ning; Tan, Yi; Li, Yonghong; Wang, Jing title: Genetic Characterization of Two Human Cases Infected with the Avian Influenza A (H5N6) Viruses — Guangxi Zhuang Autonomous Region, China, 2021 date: 2021-10-29 journal: China CDC Wkly DOI: 10.46234/ccdcw2021.199 sha: 96d755973ed3794db8e396ee03b2f71b65efa4cb doc_id: 989208 cord_uid: u5092r08 WHAT IS KNOWN ABOUT THIS TOPIC? H5N6 has replaced H5N1 as a dominant avian influenza virus (AIV) subtype in southern China. The increasing genetic diversity and geographical distribution of H5N6 pose a serious threat to the poultry industry and human health. WHAT IS ADDED BY THIS REPORT? A total of 2 cases of H5N6 that occurred from February 2021 to July 2021 in Guangxi, China were reported in this study. Phylogenetic analysis of gene was constructed, and some mutations of HA gene, PB2 gene, PA gene, M1 gene, NS1 gene, the receptor-binding site were detected. The evolutionary origins of the internal genes were different. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE? As a multi-source reassortant virus, the H5N6 highly pathogenic AIV is continuously evolving. There is an urgent need to strengthen the surveillance of drug-resistant strains and novel variants. Since the first human infections with highly pathogenic avian influenza (HPAI) H5N6 virus was detected in Sichuan Province of China in April 2014, a total of 38 human cases and 21 deaths due to H5N6 infection have been reported as of August 6, 2021 in the Western Pacific Region (1) . However, the newly emergent HPAI H5N6 virus belonging to the genetic clade 2.3.4.4 of H5 virus subtypes has possessed the capability for binding human-origin SA-α, 6 Gallinked receptor and has demonstrated more transmissibility than H5N1 virus in a ferret model (2) , suggesting that this subtype virus may be of high public health risk. Guangxi Zhuang Autonomous Region in southern China has a history of human infection with avian influenza virus (AIV). H7N9 virus emerged here (3) and HPAI H7N9 virus hit this region in 2017, with 27 infections and 14 deaths (4). We reported 2 cases infected with the H5N6 virus belonging to genetic clade 2.3.4.4b and clade 2.3.4.4h in Guangxi, China in 2021. These 2 cases occurred in two different cities. A case was admitted to the intensive care unit (ICU) due to severe clinical symptoms and was subsequently tested positive for H5N6. The other case was detected from the influenza-like illness (ILI) surveillance system. When a case is found, the local CDC immediately conducts epidemiological and environmental investigations. On February 16, 2021, a 50-year-old male (Patient A) developed a fever with chest tightness, tightness of breath, headache, cough, sputum, and pneumonia, and was transferred to the Respiratory Department of Hechi People's Hospital for hospitalization on February 21. Patient A had suffered from rheumatoid arthritis for more than 10 years, and he was found to be H5N6 influenza virus positive on February 26. He had a history of exposure to infected poultry 9 days before the onset of illness and died on March 2. On July 6, 2021, a 61-year-old female (patient B) developed a fever with a maximum temperature of 38.5 °C and was admitted to the Second People's Hospital of Guangxi for treatment on July 9. After 6 days, she was confirmed to be positive for H5N6 by Guangxi CDC. She had a history of chronic gastritis and denied having contact with live poultry. Viral RNA from the throat swabs of the two patients was extracted using the QIAamp ® Viral RNA Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Specific real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays with specific primer and probe sets for detecting avian influenza A (H5N6). The viral genomes were reversetranscribed and amplified using Easy-Fast FluA whole genome amplification one-step kit (Xinlihechuang Technology Co., Ltd.). The sequencing libraries were prepared using the Illumina Nextera ® XT Library Prep Kit. The final viral-enriched libraries were sequenced using the Illumina MiSeq platform (Illumina, San Diego, USA). The whole genome sequences of the 2 avian influenza A (H5N6) strains were assembled and obtained using CLC Genomics Workbench 9.5.2, and the 2 strains were designated A/GX-hechi/01/ 2021(H5N6) (GX01) from Patient A and A/GXguilin/11151/2021(H5N6) (GX11151) from Patient B. The genomes were aligned with the reference genomes downloaded from GISAID (www.gisaid.org) using MAFFT v7.037b (https://mafft.cbrc.jp/ alignment/software/). The genetic and evolutionary analyses were conducted in MEGA 7.0.14 (https://www. megasoftware.net/). Through sequencing and splicing, 8 gene fragments of the virus were successfully extracted. Some mutations related to viral replication, receptor-binding, mammalian virulence-related markers and drugresistance related markers were detected. The PB2 and MP genes of GX01 virus showed high homology with H9N2 viruses, and the following genes showed high homology with H5N6 viruses. The HA and NP genes of GX11151 virus showed high homology with H5N8 and H9N2 viruses, respectively, and the following genes showed high homology with H5N6 viruses (Table 1) . Phylogenetic analysis of HA gene was constructed according to the World Health Organization (WHO) reference sequence. Their nucleotide sequence showed a similarity of 90.7% between each other. The HA gene of GX01 falls into clade 2.3.4.4h, while GX11151falls into clade2.3.4.4b ( Figure 1 ). The HA cleavage site of two viruses possessed a multiple basic amino acids motif, indicating potentially high pathogenicity in chickens. The receptor-binding site at the 222-224 motif was QGG of GX01 virus and QRG of GX11151 virus, respectively, suggesting that these viruses preferred binding to avian-like receptors (α 2,3 SA) (5). However, D94N (6), S133A (7), and T156A (8) mutations in GX01 HA gene, S133A, D155N (8), T156A, and T188I (7) A263T mutation in HA gene was detected in both strains, suggesting that the virulence was enhanced. However, there was no mutation associated with resistance to NA inhibitors in the NA gene of the two strains. K389R, V598T/I mutations of PB2 gene, and N409S mutation of PA gene, which could increase virus replicative ability in mammals, were observed in both strains. Some mutations increasing virulence in A/goose/Guangdong/1/1996(H5N1) China CDC Weekly mice were detected in our strains, such as N30D, T139A, and T215A of M1 gene and P42S and 80-84 deletion of NS1 gene. The M2 gene of GX01 had the mutations of D21G and S31N, suggesting that the strain was resistant to amantadine, but GX11151 was not observed (Table 2 ). The previous study showed H5N6 has replaced H5N1 as one of the dominant AV subtypes in southern China (9) . The avian influenza A (H5N6) virus continues to threaten human life and health. I T T T T T V PB1 I368V I I I I I V Transmissible among A . Fortunately, these mutations were not found in our virus. Therefore, using NA inhibitors to treat the 2 cases infected with avian influenza A (H5N6) was still a good choice. The median age of the two cases was over 50 years old. Like other studies previously reported, the elderly people may be more vulnerable to avian influenza (3). One case was reported through ILI surveillance system (14) , indicating that the ILI system was beneficial for the detection of avian influenza cases to a certain extent. However, this may be just the tip of the iceberg, and perhaps many mild cases have not been detected. This study has certain limitations. Sampling of the environment exposed by the cases, and poultry around the living environment were missing, resulting in lacking of laboratory tests. One reason was that the period from onset to reporting was too long for sampling, in spite of dead poultry found in epidemiology survey, and another reason was that patient B had no clear history of exposure to poultry. At present, coronavirus disease (COVID-19) has caused a worldwide pandemic (15), and over 200 million people have been infected. Meanwhile, the increasing trend of human infection with avian influenza virus has become an important public health issue that cannot be ignored, alerting us that COVID-19 and avian influenza may be simultaneously prevalent in some regions. 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