Novel #Reassortant #Avian #Influenza A(#H5N6) Viruses in #Humans, #Guangdong, #China, 2015 (@CDC_EIDjournal, edited)

[Source: US Centers for Disease Control and Prevention (CDC), Emerging Infectious Diseases Journal, full page: (LINK). Edited.

Volume 22, Number 8—August 2016 / Letter

Novel Reassortant Avian Influenza A(H5N6) Viruses in Humans, Guangdong, China, 2015


To the Editor:

Avian influenza A(H5N6) influenza viruses have circulated among poultry in southern (Jiangxi, Guangdong) and western (Sichuan) provinces of China since 2013 (1,2). In 2014, outbreaks of H5N6 virus infection occurred among poultry in China, Laos, and Vietnam (1). In April 2014, the first case of highly pathogenic H5N6 infection among humans was detected in Sichuan Province (3); the second case was detected on December 3, 2014, in Guangdong Province (4). In December 2015, 4 humans in Guangdong Province were infected with H5N6 influenza (5,6).

To study the genetic basis of continuing human infections with this avian influenza subtype, we sequenced the complete genomes of 2 of the 4 human H5N6 isolates obtained in December 2015 in Guangdong Province. We compared these sequences with those of 1 H6N6 and 8 H5N6 influenza viruses isolated from birds in live poultry markets in this region during 2013–2015 (Technical Appendix[PDF – 1.63 MB – 12 pages]) and other published genomes of H5, H6N6, and H9N2 avian influenza viruses (Technical Appendix). Phylogenetic analyses of the hemagglutinin (HA) genes showed that all human H5N6 isolates belonged to clade (Technical Appendix Figure 1, panel A). HA and neuraminidase (NA) genes of some H5N6 viruses isolated in Guangdong Province during 2013–2014 were in the Sichuan-like lineage, but all of those from 2015 were in the Jiangxi-like lineage (Technical Appendix Figure 1, panels A, B).

Despite the similarities of the HA and NA genes, the 6 internal genes from the 2 human isolates from 2015, A/Guangdong/ZQ874/2015 (H5N6) and A/Guangdong/SZ872/2015 (H5N6) were different from 2 human H5N6 isolates from 2014, A/Sichuan/26221/2014 (H5N6) and A/Guangzhou/39715/2014 (H5N6).

The polymerase basic (PB) 2 gene from isolate A/Guangdong/ZQ874/2015 (H5N6) appears to have derived from an H6N6 virus isolated from a duck; all other genes in this isolate were derived from H5N6 viruses that have been circulating among poultry since 2013 (Technical Appendix Figure 1, panel C; Technical Appendix Figure 2).

This isolate showed high nucleotide identity to 6 of the 8 genes (HA, 96.5%; NA, 98.2%; nucleoprotein (NP), 98.5%; polymerase acidic (PA), 98.3%; PB1, 98.1%; PB2, 98.4%) of the isolate A/chicken/Guandong/FG594/2015 (H5N6); the identities of the matrix (M) and nonstructural protein (NSP) genes were 76.2% and 79.8% similar, respectively. This finding suggests that undetected reassortants of H5N6 may exist.

The other human isolate, A/Guangdong/SZ872/2015 (H5N6), showed high nucleotide identity with A/Yunnan/0127/2015 (H5N6), an isolate collected from a person in Yunnan Province (GenBank accession nos. KT963053–60; Technical Appendix Table), for all 8 genes (HA, 97.2%; M, 97.7%; NA, 96.8%; NP, 98.3%; NSP, 93.2%; PA, 95.9%; PB1, 96.9%; PB2, 94.0%).

The 6 internal genes of A/Guangdong/SZ872/2015 (H5N6) appear to have come from the enzootic H9N2 (ZJ-HJ/07) virus lineage (Technical Appendix Figure 1, panel C). These findings show that the circulating H5N6 virus in southern China has reassorted with enzootic H6N6 and H9N2 viruses, resulting in new H5N6 viruses that are capable of infecting humans.

We compared the 2 newly sequenced genomes with 3 available genomes of human influenza virus strains in public databases to determine if they had attained key molecular features associated with increased virulence in mammals, mammalian transmissibility, and antiviral resistance (Table).

The HA gene cleavage site encoded by all 5 isolates from humans showed a multiple basic amino acid motif (REKRRKR↓G), which is indicative of high pathogenicity in poultry.

The viruses isolated from humans in 2014 had no mutations associated with reduced sensitivity to adamantine antiviral drugs, but 2 of the 3 viruses isolated in 2015 have the 31N amino acid in M2, suggesting that those 2 viruses have acquired resistance. Thus, this virus lineage could be a great threat to public health.

Although H9N2 is not highly pathogenic in poultry, it provides internal genes for the recent emergence of many novel avian influenza viruses that infect humans, such as the H5N6 virus in this study, as well as the H7N9 (7,8) and H10N8 (9) viruses. Infection with H6 subtype viruses results in no clinically significant signs of disease in poultry (10), but co-circulation of these viruses with other subtypes among poultry results in transfer of internal genes. This reassortment has resulted in a major increase in genetic diversity among the H5N6 viruses that cause human infections; therefore, low-pathogenicity viruses in poultry should also be controlled in poultry.

In summary, we isolated 2 novel reassortant H5N6 viruses from 2 patients in Guangdong Province, China. The internal genes of these strains are different from those found in the first wave of H5N6 infections in 2014.

The PB2 of 2 human isolate A/Guangdong/ZQ874/2015 (H5N6) appears to have been derived from a duck H6N6 virus, and all other genes of this virus originated in circulating H5N6 viruses.

In contrast, the 6 internal genes of the other human isolate, A/Guangdong/SZ872/2015 (H5N6), were derived from enzootic H9N2 viruses. Although human infection has been sporadic, the co-circulation and reassortment of this virus with other enzootic low pathogenicity influenza viruses has resulted in new reassortant viruses. Further surveillance of birds is needed to monitor the spread of this novel virus.


Yong-Yi Shen1, Chang-Wen Ke1, Qian Li1, Run-Yu Yuan, Dan Xiang, Wei-Xin Jia, Yun-Di Yu, Lu Liu, Can Huang, Wen-Bao Qi, Reina Sikkema, Jie Wu, Marion Koopmans, and Ming Liao

Author affiliations: Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, Guangzhou (Y.-Y. Shen); College of Veterinary Medicine, South China Agricultural University, Guangzhou, China (Y.-Y. Shen, Q. Li, W.-X. Jia, Y.-D. Yu, W.-B. Qi, M. Liao); Guangdong Provincial Center for Disease Control and Prevention, Guangzhou (C.-W. Ke, R.-Y. Yuan, J. Wu); Shantou University Medical College, Shantou, China (D. Xiang, L. Liu, C. Huang); Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture, Guangzhou (W.-B. Qi); National Institute of Public Health and the Environment, Bilthoven, the Netherlands (R. Sikkema, M. Koopmans); National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangzhou (M. Liao)



This study was supported by the National Natural Science Foundation of China (U1501212); Guangdong Natural Science Funds for Distinguished Young Scholar (2014A030306046), was a Key Project of the Agricultural Ministry (CARS-42-G09), and the Modern Agriculture Talents Support Program (2012, no. 160).



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Suggested citation for this article: Shen YY, Ke CW, Li Q, Yuan RY, Xiang D, Jia WX, et al. Novel reassortant avian influenza A(H5N6) viruses in humans, Guangdong, China, 2015 [letter]. Emerg Infect Dis. 2016 Aug [date cited].

DOI: 10.3201/eid2208.160146

1These authors contributed equally to this article.

Keywords: Research; Abstracts; H5N6; H6N6; H9N2; Avian Influenza; Human; China; Guangdong; Reassortant Strain.


Highly pathogenic #avian #influenza #H5N6 viruses exhibit enhanced #affinity for #human type #sialic acid #receptor and in-contact #transmission in model #ferrets (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

Highly pathogenic avian influenza H5N6 viruses exhibit enhanced affinity for human type sialic acid receptor and in-contact transmission in model ferrets

Honglei Sun a, Juan Pu a, YanDi Wei a, Yipeng Sun a, Jiao Hu b, Litao Liu a, Guanlong Xu a, Weihua Gao a, Chong Li a, Xuxiao Zhang a, Yinhua Huang a, Kin-Chow Chang c, Xiufan Liu b and Jinhua Liu a⇑

Author Affiliations: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China[a]; Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, China[b]; School of Veterinary Medicine and Science, University of Nottingham Sutton Bonington Campus, Sutton Bonington, UK[c]



Since May 2014, highly pathogenic avian influenza (HPAI) H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals are not known. We characterized the virus receptor binding affinity, pathogenicity and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired binding affinity for human-like SAα2,6Gal linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less severe pathogenicity in comparison with their parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans.



Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel clade of H5 virus subtypes including H5N2, H5N6 and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals are not known. Here, we found that natural avian H5N6 viruses have acquired high affinity for human-type virus receptor. In comparison with parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets, but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

FOOTNOTES: Correspondence and requests for materials should be addressed to J.L. (

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Keywords: Research; Abstracts; H5N6; Avian Influenza.


The #molecular #characterizations of surface #proteins #hemagglutinin and #neuraminidase from recent #H5Nx #avian #influenza #viruses (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

The molecular characterizations of surface proteins hemagglutinin and neuraminidase from recent H5Nx avian influenza viruses

Hua Yang 1, Paul J. Carney 1, Vasiliy P. Mishin 1, Zhu Guo 1, Jessie C. Chang 1, David E. Wentworth 1, Larisa V. Gubareva 1 and James Stevens 1*

Author Affiliations: 1Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30329



During 2014, a subclade HPAI A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015 the virus was detected in wild birds in Canada and the U.S. and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North America lineages. In particular, viruses were found with N1, N2 and N8 neuraminidase vRNAs, and are collectively referred to as H5Nx viruses. In the U. S., more than 48 million domestic birds have been affected. Here, we present a detailed structural and biochemical analysis of the surface antigens from H5N1, H5N2 and H5N8 in addition to a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses, and to continually assess future changes that may increase their pandemic potential.



The H5Nx viruses emerging in North America, Europe, and Asia are of great public health concern. Herein, we report a molecular and structural study of the major surface proteins from several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preference and susceptibility to antivirals, which is central to pandemic risk assessment.



*Corresponding author: James Stevens, PhD, Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, 1600 Clifton Road – Mail Stop D-30, Atlanta, GA 30333, Phone: (404) 639-5008, Email:

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Keywords: Research; Abstracts; H5N1; H5N2; H5N6; H5N8; Avian Influenza.


#Emergence and #dissemination of clade #H5Nx #influenza viruses-how is the Asian HPAI H5 lineage maintained (Curr Opin Virol., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Curr Opin Virol. 2016 Mar 15;16:158-163. doi: 10.1016/j.coviro.2016.02.005. [Epub ahead of print]

Emergence and dissemination of clade H5Nx influenza viruses-how is the Asian HPAI H5 lineage maintained.

Claes F1, Morzaria SP2, Donis RO3.

Author information: 1Food and Agriculture Organization of the United Nations (FAO), Regional Office for Asia and the Pacific, Bangkok, Thailand. Electronic address: 2Food and Agriculture Organization of the United Nations (FAO), Viale delle Terme Di Caracalla, Rome 00153, Italy. 3Influenza Division, Centers for Disease Control and Prevention, USA. Electronic address:



Highly pathogenic avian influenza (HPAI) A(H5N1) viruses containing the A/goose/Guangdong/96-like (GD/96) HA genes circulated in birds from four continents in the course of 2015 (Jan to Sept). A new HA clade, termed, emerged around 2010-2011 in China and revealed a novel propensity to reassort with NA subtypes other than N1, unlike dozens of earlier clades. Two subtypes, H5N6 and H5N8, have spread to countries in Asia (H5N6), Europe and North America (H5N8). Infections by clade viruses are characterized by low virulence in poultry and some wild birds, contributing to wide geographical dissemination of the viruses via poultry trade and wild bird migration.

Copyright © 2016. Published by Elsevier B.V.

PMID: 26991931 [PubMed – as supplied by publisher]

Keywords: Research; Abstracts; Avian Influenza; H5N1; H5N6; H5N8.


#Detection of highly pathogenic #zoonotic #influenza #virus #H5N6 by RT-q #PCR (Virol J., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Virol J. 2015 Feb 8;12:18. doi: 10.1186/s12985-015-0250-3.

Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction. [      ]

Heine HG1, Foord AJ2, Wang J3, Valdeter S4, Walker S5, Morrissy C6, Wong FY7, Meehan B8.

Author information: 1CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 2CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 3CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 4CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 5CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 6CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 7CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. 8CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia.




Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade (previously provisionally referred to as clade, and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms.


Fluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes.


Laos influenza A(H5N6) virus representative of proposed clade, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.

PMID: 25889293 [PubMed – indexed for MEDLINE]

PMCID: PMC4328077 / Free PMC Article

Keywords: Research; Abstracts; Avian Influenza; H5N6; Diagnostic Tests.