Five Independent Cases of Human Infection with Avian Influenza H5N6

China CDC Weekly

Outbreak Reports

Five Independent Cases of Human Infection with Avian Influenza H5N6 — Sichuan Province, China, 2021

Chongkun Xiao1,&; Jianan Xu1,&; Yu Lan2,&; Zhongping Huang1; Lijun Zhou1; Yaxin Guo2; Xiyan Li2; Lei Yang2; George F. Gao2; Dayan Wang2; William J. Liu2,#; Xingyu Zhou1,#; Huiping Yang1,#

including 21 deaths. This year, 10 sporadic human Summary infections have been recorded in Sichuan Province, What is known about this topic? Anhui Province, Guangxi Zhuang Autonomous

The emerging H5Ny lineages of the avian influenza Region, and Chongqing Municipality ( 8). virus (AIV) with genomic reassortments have posed a continuous threat to animals and human beings. Since INVESTIGATION AND RESULTS the first case of avian influenza A (H5N6) virus infection in 2014, the World Health Organization has Herein, we report the 5 cases infected by AIV H5N6 reported a total of 38 cases by August 6, 2021. in Sichuan Province, China in 2021. These 5 What is added by this report? independent cases occurred in 5 different districts or A total of 5 new cases of H5N6 that occurred from counties from 4 cities (Figure 1A, i.e., Jinjiang District May 2021 to July 2021 in Sichuan Province, China of Chengdu City, Kaijiang County and Xuanhan were reported in this study. Epidemiological and County of Dazhou City, Bazhou District of Bazhong laboratory information of the five cases were City, and Nanxi District of Yibin City). All four cities investigated. The genomic analysis of the H5N6 are located in the east of Sichuan Province, China genomes showed the features of AIV genomic (Figure 1A). A case was sampled by nasopharyngeal reassortments and key residue substitutions. swabs and sent to the laboratory for quantitative What are the implications for public health polymerase chain reaction (qPCR) testing. The positive practice? results will be sequenced and sent to China CDC for The emergence of human cases infected by AIV H5Ny virus isolation. When a case was found, the local CDC lineages through time demonstrates a risk of the and the municipal and/or provincial CDCs form two persistence and evolution of the virus to trigger or three levels of investigation groups to carry out the sporadic outbreaks and even pandemics, which need epidemiological investigation of avian influenza cases. continuous surveillance. The timeline and investigation group for each case were shown in Table 1.

As a highly contagious virus, the avian influenza Patient 1 in Jinjiang District of Chengdu City was a virus (AIV) circulates among birds as its reservoir host 49-year-old (y) female who developed headache and (1). The spillover to human beings and other animal nasal stuffiness on May 13. On May 26, the respiratory species occurs frequently, leading to cross-species tract sample was tested for influenza viruses in Huaxi Hospital and was found to be H5N6 influenza virus infection that may trigger mild outbreaks and even positive, and on the next day, Chengdu Municipal pandemics (2). In recent years, AIV H5Ny lineages CDC and Sichuan Provincial CDC confirmed the (i.e., H5N1, H5N2, H5N6, and H5N8) have proved laboratory test as positive for H5N6 (Figure 1B and the capacity for zoonotic spread and genomic Table 1). The patient (Patient 2) from Kaijiang reassortments amongst this viral group and thus pose a County of Dazhou City, who was a 57y male, had severe threat to animals and human beings (3–4). symptom onset on June 22 with cough and asthma. Among these AIVs, H5N6 was first detected in 1975 The patient was laboratory confirmed as H5N6 (5), and the first reported case of human infection with infection by Sichuan CDC on July 8. Patient 3 was a a novel H5N6 was dated in 2014 (6–7). By August 51y female, from Xuanhan County of Dazhou City, 2021, the World Health Organization (WHO) that had a headache on June 25. Through qPCR reported a total of 38 laboratory-confirmed cases of testing of alveolar lavage fluid specimens, the case was human infection with influenza A(H5N6) virus, laboratory diagnosed as H5N6 infection by Sichuan

Chinese Center for Disease Control and Prevention CCDC Weekly / Vol. 3 / No. 36 751 China CDC Weekly

A N

③ Xuanhan, ④ Bazhou, Dazhou Bazhong ① Jinjiang, Chengdu ② Kaijiang, N Dazhou ⑤ Nanxi, Yibin

0 140 280 miles

B Case 1 Case 3 Case 5

2021.5.13 2021.6.25 2021.7.8 Female Female Female 49y 51y 65y Jinjiang, Chengdu Xuanhan, Dazhou Nanxi, Yibin May June July 2021.6.22 2021.6.30 Male Male 57y 55y Kaijiang, Dazhou Bazhou, Bazhong

Case 2 Case 4

FIGURE 1. Temporal and spatial distribution of human infections with avian influenza A(H5N6) viruses in Sichuan Province, China, May to July 2021. (A) Geographical distribution of H5N6 virus infection among humans*; (B) The timeline and the basic demographic information of the H5N6 human cases. Abbreviation: y=years old. * The sequence number of case-patients in place is based on the disease onset date. CDC on July 7. Patient 4 was a 55y male in Bazhou baby poult before symptom onset. Patients 4 and 5 had District of Bazhong City. He developed initial no history of visiting LPMs 4 weeks before their illness symptoms of fever on June 30 and was laboratory onset. The poultry farmed by Patient 5 were usually confirmed as H5N6 infection on July 6. Patient 5, sent to the adjacent pond for breeding where wild birds female, 65y, from Nanxi District of Yibin City had (predominantly, white cranes) were frequently symptom onset on July 8. The pharyngeal swab from observed. There were dead poultry that were farmed by Patient 5 was laboratory tested as H5N6 positive on Patients 2, 3, 4, and 5, and they had contact with or July 20. ate these dead poultry before their symptom onset The epidemiological investigation found that all 5 (Table 1). patients had been exposed to live poultry before their In light of laboratory testing of samples collected disease onset (Table 1). Patient 1 bought duck from a from the poultry market contacted by Patient 1, H5N6 poultry market on May 5, i.e., 8 days before the were qPCR detected positive. The poultry feeding symptom onset. Patients 2, 3, 4, and 5 lived in rural settings of Patient 2–5 were tested, with positive results areas and raised chickens, ducks, and gooses in their in the environment samples related to all four patients backyard for self-consumption. Patients 2 and 3 had (Supplementary Table S1, available in http://weekly. visited the live poultry markets (LPMs) and purchased chinacdc.cn/).

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All five patients had a history of contact with birds, Jiangxi, China in 2016, compared to the NS of so the following measures were taken. 1) Ten days of Dazhou/2021 (Table 2). The maximum likelihood health monitoring was implemented of close contacts (ML) phylogenies tree [root at and possible exposed personnel of all patients. 2) A/Goose/Guangdong/1/96(H5N1)] was generated by Poultry and environmental disposal were carried out in RAxML (version 8.2) program (9). The genomes of affected areas. The patients’ families and the patients’ Dazhou/2021 and Bazhong/2021 have a similarity neighbors’ families carried out poultry culling and >99% for all the 6 available segments. The molecular disposal, and thoroughly disinfected bird-related phylogenetic analysis showed that the HA genes of environments. 3) Strengthened management of poultry both Dazhou/2021 and Bazhong/2021 belong to the breeding sites and live poultry trading markets. This 2.3.4.4b and have the highest homology with involved comprehensive rectification, cleaning, and A/chicken/Omsk/0112/2020 (A/H5N8) from Omsk disinfection of the live poultry trading market in the Russia (Figure 2). The genomes of Dazhou/2021 and district and county. 4) Influenza-like illness (ILI) Bazhong/2021 have a similarity >99% for all the 6 surveillance was strengthened in affected districts and available segments. The molecular phylogenetic counties. For outpatient and emergency patients who analysis showed that the HA genes of both met the definition of ILI and patients with severe acute Dazhou/2021 and Bazhong/2021 belong to the respiratory tract infection, the history of poultry 2.3.4.4b and have the highest homology with exposure was inquired for and respiratory tract samples A/chicken/Omsk/0112/2020 (A/H5N8) from Omsk were collected for testing. During the strengthened Russia (Figure 2). surveillance period, the positive rate of ILI had a mild The molecular characterization of the Dazhou/2021 upward trend, but these ILI were all influenza B and Bazhong/2021 strains were analyzed and related viruses. Influenza A viruses had not been isolated key sites in all H5N6 were analyzed at the same time (except for one case of H9N2) in all of Sichuan (Supplementary Table S2, available in http://weekly. Province, and there was no A (subtype not chinacdc.cn/). The cleavage site of HA protein determined). 5) Strengthened health education by possessed a multiple basic amino acids motif carrying out publicity and education on avian influenza (LREKRRKR↓G), which indicated high prevention and control knowledge throughout the pathogenicity to chickens. The amino acid of the NS1 province, improving the disease prevention awareness protein of these 2 strains at position 92 was aspartic of professional people, and reducing the risk of human acid, However, most H5N6 strains (n=1,329) have the infection with avian influenza virus. 92E mutation; this D92E mutation has been correlated The full genome of the virus from Patient 3 with increased virulence and/or cytokine resistance (A/Sichuan/06681/2021(H5N6), Dazhou/2021) and 6 (10). NA protein was deleted at the stalk region gene segments except for PB2 and PA of the virus from (position 59–69), and no oseltamivir-associated Patient 4 (A/SiChuan-Bazhong/1/2021(H5N6), resistance mutations in amino acid residues were Bazhong/2021) were successfully sequenced. The found. Host-specific related sites, such as receptor nucleotide sequences similarity of 8 segments of binding Q226L of HA fragment and E627K of PB2 Dazhou/2021 were analyzed with the online Basic fragment were not found, which indicated that both Local Alignment Search Tool (BLAST) (Table 2), and Dazhou/2021 and Bazhong/2021 strains still possess the PB2 segment was found to have similarity with features of avian origin. chicken H3N2 strain from Guangxi, China (96%) in 2014; PB1 and PA genes were 97%–98% similarity to DISCUSSION that of H5N6 strains from poultry environment samples in Guangdong in 2017–2018 season. NA and The AIV H5N6 was first reported as a low- NP segments were highly similarity to human infecting pathogenic AIV (LPAIV) decades ago (5). Since the H5N6 strains from Anhui Province in 2020 (99%). strain a/gs/gd/1/96 (H5N1) appeared in the 1990s, The MP segment has a high similarity with chicken outbreaks of highly pathogenic AIV (HPAI) have H5N8 strain from Kostroma, Russia in 2020 and the occurred frequently. Before 2010 there was no NS segment was highly similar to chicken H3N2 strain evidence of reassortment of the H5N1 viruses with NA from Ganzhou, China in 2016. Interestingly, for strain subtypes other than N1 (11). However, H5 reassorted Bazhong/2021 in our study, only the NS segment has a with different specific NA subtypes, termed H5N2, different closest stain i.e., H4N2 and H3N2 from H5N3, H5N5, H5N6, H5N8 after 2010 (12). These

Chinese Center for Disease Control and Prevention CCDC Weekly / Vol. 3 / No. 36 753 754 TABLE 1. Demography and epidemiological profiles of the 5 cases in Sichuan. Age of Date of Consumption Date of Date of Date of Contact with Date of used Date of Investigation Case No. Region case/years laboratory of dead Outcome onset hospitalization visited LPM dead poultry Tamiflu Investigation group old confirmation poultry Sichuan, CCDC Weekly/Vol. 3/No.36 Jinjiang, 1 49 2021-05-13 2021-05-16 2021-05-26 2021-05-05 Unknown No 2021-05-27 Death 2021-05-26 Chengdu and Chengdu Jinjiang CDC Sichuan, Kaijiang, 2 57 2021-06-22 2021-07-05 2021-07-08 2021-06-13 Yes Yes 2021-07-08 Death 2021-07-08 Dazhou and Dazhou Kaijiang CDC Sichuan, Xuanhan, 3 51 2021-06-25 2021-07-02 2021-07-07 2021-06-04 Yes Unknown Not used Death 2021-07-06 Dazhou and Dazhou Xuanhan CDC Sichuan, Bazhou, 4 55 2021-06-30 2021-07-04 2021-07-06 Unknown Yes No 2021-07-05 Recovered 2021-07-05 Bazhong and Bazhong Bazhou CDC Yibin and 5 Nanxi, Yibin 65 2021-07-08 2021-07-13 2021-07-20 Unknown Yes Yes 2021-07-19 Recovered 2021-07-18 Nanxi CDC Abbreviation: LPM=live poultry market. China CDCWeekly TABLE 2. Similarity of 8 segments of the viruses analyzed by online basic local alignment search tool. Segments Bazhong/2021 Identity/Length (%) Dazhou/2021 Identity/Length (%) Collection location A/chicken/Guangxi/165C7/2014 (A/H3N2) PB2 No sequence detected Guangxi, China 2,263/2,342 (96%) A/Env/Guangdong/zhanjiang/C18277136/2018-04-02 (A/H5N6) A/Env/Guangdong/zhanjiang/C18277136/2018-04-02 (A/H5N6) PB1 Guangdong, China 2,304/2,344 (98%) 2,310/2,345 (98%) A/Env/Guangdong/zhanjiang/C17277346/2017-12-05 (A/H5N6) PA No sequence detected Guangdong, China Chinese Centerfor DiseaseControlandPrevention 2,184/2,233 (97%) A/chicken/Omsk/0112/2020 (A/H5N8) A/chicken/Omsk/0112/2020 (A/H5N8) HA Omsk, Russia 1,766/1,776 (99%) 1,760/1,772 (99%) A/Anhui/2021-00011/2020 (A/H5N6) A/Anhui/2021-00011/2020 (A/H5N6) NP Anhui, China 1,552/1,565 (99%) 1,555/1,564 (99%) A/Anhui/2021-00011/2020 (A/H5N6) A/Anhui/2021-00011/2020 (A/H5N6) NA Anhui, China 1,422/1,432 (99%) 1,423/1,431 (99%) A/chicken/Kostroma/304-10/2020 (A/H5N8) A/chicken/Kostroma/304-10/2020 (A/H5N8) MP Kostroma, Russia 1,025/1,027 (99%) 1,026/1,027 (99%) A/Environment/Jiangxi/47054/2016 (A/H4N2) A/chicken/Ganzhou/GZ43/2016 (A/H3N2) NS Jiangxi, China 870/891 (97%) 873/890 (98%) China CDC Weekly

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FIGURE 2. Maximum likelihood phylogenetic relationships of H5 viruses’ hemagglutinin (HA) genes. Note: Phylogenetic included most of H5 clades (names were showed at leaves tip) and rooted to A/Goose/Guangdong/1/96(H5N1), constructed with 1,000 bootstrap replicates (above branches). The two stars indicate the strains of this study. new virus subtypes emerged through multiple genetic including genomic epidemiology, is necessary on wild reassortments of different subtype viruses within birds, poultry, and the environment. resident domestic and wild bird populations and The novel clade 2.3.4.4 H5N6 that was first continued to circulate in domestic poultry populations, reported in 2013, then reported in Laos and Vietnam leading to wider geographical spread and raising great in 2014/2015, with evidence of sustained transmission concern based on their pandemic potential (13). With and further geographical spread in both countries. the cross-infection of wild birds and poultry, the Later on, a series of poultry outbreaks in Japan, epidemic of viruses in poultry has accelerated. To date, Myanmar, and the Republic of Korea have been found a total of 38 laboratory-confirmed cases of human to be related by H5N6. By 2017, it began spreading to infection with influenza A (H5N6) virus, including 21 some European countries such as Greece, Germany, deaths. Our study found that all the 5 patients had the Netherlands, and Switzerland (14). Due to the been exposed to live poultry before their disease onset. genetic diversity of the virus, the World Health Given this, more research, enhanced surveillance, Organization (WHO) subdivided 2.3.4.4 into a–h

Chinese Center for Disease Control and Prevention CCDC Weekly / Vol. 3 / No. 36 755 China CDC Weekly clades, a total of 8 new clades. Even if it is so finely (6181):235. http://dx.doi.org/10.1126/science.1254664.

2. Liu WJ, Wu Y, Bi YH, Shi WF, Wang DY, Shi Y, et al. Emerging divided, recent serological studies have found that the HxNy influenza a viruses. Cold Spring Harb Perspect Med 2020 vaccine strains recommended by WHO cannot fully a038406. http://dx.doi.org/10.1101/cshperspect.a038406. cover the emerging strains in these clades (15). Thus, 3. Shi WF, Gao GF. Emerging H5N8 avian influenza viruses. Science 2021;372(6544):784 − 6. http://dx.doi.org/10.1126/science.abg6302.

the balance between viral evolution and the capacity of 4. Quan CS, Wang QL, Zhang J, Zhao M, Dai QG, Huang T, et al. the candidate vaccine protection needs to be timely Avian influenza A viruses among occupationally exposed populations, updated which will reduce infection events and China, 2014-2016. Emerg Infect Dis 2019;25(12):2215 − 25. http://dx.doi.org/10.3201/eid2512.190261.

mortality in humans and animals. 5. Bi YH, Kun M, Shi WF, Liu D, Yu XL, Gao ZM, et al. Two novel Fortunately, there is no evidence of sustainable reassortants of avian influenza A (H5N6) virus in China.. J Gen Virol human-to-human transmission of H5N6, considering 2015;96(Pt 5):975-981. http://dx.doi.org/10.1099/vir.0.000056. 6. Zhang RS, Chen TM, Ou XH, Liu RC, Yang Y, Ye W, et al. Clinical, that all cases in this study were independent and have epidemiological and virological characteristics of the first detected no epidemiological correlation. A one-health approach human case of avian influenza A(H5N6) virus. Infect Genet Evol needs to be strengthened to trace the source of 2016;40:236 − 42. http://dx.doi.org/10.1016/j.meegid.2016.03.010. 7. Pan M, Gao RB, Lv Q, Huang SH, Zhou ZH, Yang L, et al. Human infection in time, block the cross-species spread of the infection with a novel, highly pathogenic avian influenza A (H5N6) virus: virological and clinical findings. J Infect 2016;72(1):52 − 9. virus, reduce risks, and protect people ’s health. http://dx.doi.org/10.1016/j.jinf.2015.06.009.

Acknowledgments: Ms. Jie Zhang, Mr. Idrissa 8. WHO. Avian influenza weekly update number 804. https://www.who. Kamara, and Dr. Chuansong Quan. int/docs/default-source/wpro---documents/emergency/surveillance/ Funding: Supported by the National Natural avian-influenza/ai_20210806.pdf?Status=Master&sfvrsn=5f006f99_42. [2021-8-6].

Science Foundation of China (No.82041033), Sichuan 9. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and Science and Technology Program (2020FYS0015; post-analysis of large phylogenies. Bioinformatics 2014;30(9):1312 − 3. 2020FYS0017). W.J.L. supported by the Excellent http://dx.doi.org/10.1093/bioinformatics/btu033. 10. Li MY, Wang BH. Homology modeling and examination of the effect Young Scientist Program of the NSFC (No. of the D92E mutation on the H5N1 nonstructural protein NS1 81822040). effector domain. J Mol Model 2007;13(12):1237 − 44. http://dx.doi.org/ 10.1007/s00894-007-0245-0. doi: 10.46234/ccdcw2021.187 11. Liu D, Liu XL, Yan JH, Liu WJ, Gao GF. Interspecies transmission and host restriction of avian H5N1 influenza virus. Sci China C Life Sci # Corresponding authors: William J. Liu, [email protected]; 2009;52(5):428 − 38. http://dx.doi.org/10.1007/s11427-009-0062-z. Xingyu Zhou, [email protected]; Huiping Yang, lydia_ping106@

12. Smith GJD, Donis RO. Nomenclature updates resulting from the 163.com. evolution of avian influenza A(H5) virus clades 2.1.3.2a, 2.2.1, and 1 2.3.4 during 2013-2014. Influenza Other Respir Viruses 2015;9(5): Sichuan Center for Disease Control and Prevention, Chengdu, 271 − 6. http://dx.doi.org/10.1111/irv.12324. Sichuan, China; 2 NHC Key Laboratory of Biosafety, National 13. Bi YH, Li J, Li SQ, Fu GH, Jin T, Zhang C, et al. Dominant subtype Institute for Viral Disease Control and Prevention, Chinese Center for switch in avian influenza viruses during 2016-2019 in China. Nat Disease Control and Prevention, Beijing, China. & Commun 2020;11(1):5909. http://dx.doi.org/10.1038/s41467-020- Joint first authors. 19671-3.

14. Bi YH, Chen QJ, Wang QL, Chen JJ, Jin T, Wong G, et al. Genesis, Submitted: August 18, 2021; Accepted: August 31, 2021 evolution and prevalence of H5N6 avian influenza viruses in China. Cell Host Microbe 2016;20(6):810 − 21. http://dx.doi.org/10.1016/j. REFERENCES chom.2016.10.022. 15. Chen JH, Li X, Xu LY, Xie SM, Jia WX. Health threats from increased antigenicity changes in H5N6-dominant subtypes, 2020 China. J Infect

1. Gao GF. Influenza and the live poultry trade. Science 2014;344 2021;83(2):e9 − 11. http://dx.doi.org/10.1016/j.jinf.2021.06.013.

756 CCDC Weekly / Vol. 3 / No. 36 Chinese Center for Disease Control and Prevention China CDC Weekly – + + + + environment qPCR of living * + + + ND ND qPCR of upstream supplier environment Phenotypic effect Altered virulence in mice + + + + + Antiviral amantadine resistance Virulence increases in chickens Increases pathogenicity in mice Promotes viral replication in mice environment qPCR of poultry Enhances viral replication in mammalian cells in vitro Reduces the susceptibility of neuraminidase inhibitors Associates with increased virulence of AIVs in mammals Correlated with increased virulence and/or cytokine resistance Chicken The substitution at residue 158 leads to a loss of glycan chain modification in the 150-loop, which avoids the potential steric hindrance for binding human receptors. Critical for binding the α-2,6-linked receptor and enabling transmission in mammals Chicken, duck Chicken, duck Chicken, duck Poultry species of upstream supplier Not purchased in 2021 * 1455

I 1 S 5 Y 2 V 2 D 4 H 1 N 3 G 1 M 4 V 37 K 18 L 367 D 355 T 1648 E 1673 P 1681 E 1329 N 1803 H 1744 D 1689 Q 1813 Q 1693 All H5N6 QRETRG 10 L-I-A-S-G 10 L-G-A-N-G 6 L-A-A-S-G 16 L-V-A-N-G 165 L-V-A-S-G LKERRRKRG 29 SRERRRKRG 47 LREKRRKRG 305 LRERRRKRG 1388 s chicken and duck farm ' Not purchased in 2021 Chenghua district market Yunchengzhai chicken farm Luo T E P N H D D Q Q Specify the upstream supplier Private vendors in Wangjia town L-V-A-S-G Dazhou/2021 LREKRRKRG Chicken T E P N H D D Q Q Chicken, duck Chicken, duck Chicken, duck Poultry species L-V-A-S-G Chicken, duck, goose LREKRRKRG Bazhong/2021 Market poultry Domestic Domestic Domestic Domestic Source of D92E T271A Q226L E627K P212S H274Y Q591K N158D D701N AA position Cleavage site Region Nanxi, Yibin Kaijiang, Dazhou Bazhou, Bazhong Jinjiang, Chengdu Xuanhan, Dazhou L26F, V27A, A30T, S31N, G34E 1 2 3 4 5 No. HA NA M2 PB2 NS1 Case * * Genes SUPPLEMENTARY TABLE S1. Poultry environment investigation and testing. ND: Not done. SUPPLEMENTARY TABLE S2. The molecular characteristics of the H5N6 influenza viruses isolated from Sichuan Province. Note: Bold is consistent with the results of this study. Only show top 5, if the candidates are more than 5.

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