Wu H et al 2016

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Virus Genes (2016) 52:863–866 DOI 10.1007/s11262-016-1368-0

Genetic and molecular characterization of a novel reassortant H2N8 subtype avian influenza virus isolated from a domestic duck in Zhejiang Province in China Haibo Wu1 • Xiuming Peng1 • Xiaorong Peng1 • Linfang Cheng1 • Nanping Wu1

Received: 16 April 2016 / Accepted: 22 June 2016 / Published online: 5 July 2016 Ó Springer Science+Business Media New York 2016

Abstract The circulation of the H2 subtype influenza viruses in domestic animals increases the risk of human exposure to these viruses. An H2N8 avian influenza virus (AIV) was isolated from a domestic duck during AIV surveillance of poultry in live poultry markets (LPMs) in Zhejiang Province, Eastern China, in 2013. The phylogenetic trees suggested that this strain is a novel reassortant virus derived from multiple AIV subtypes from aquatic birds and poultry in Eastern Asia. Although this reassortant strain exhibited low pathogenicity in mice, it was able to replicate in the lungs of the mice without prior adaptation. Continued surveillance of domestic ducks in LPMs is required for early detection of AIV outbreaks in poultry and humans. Keywords Avian influenza virus  H2N8  Genetic analysis  Virus reassortment  Domestic ducks

Edited by William Dundon. Haibo Wu and Xiuming Peng have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11262-016-1368-0) contains supplementary material, which is available to authorized users. & Nanping Wu [email protected] Haibo Wu [email protected] 1

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, Zhejiang, China

Introduction Among the 18 hemagglutinin (HA) subtypes, only H1, H2, and H3 subtype influenza viruses have caused human pandemics. The H2N2 influenza virus that caused the 1957 Asian pandemic, resulting in almost two million deaths, derived its genes from human H1N1 and avian H2N2 viruses [1]. The H2 subtype influenza viruses have not circulated in humans since 1968; consequently, individuals born subsequently lack humoral immunity to H2 viruses and are susceptible to infection [2]. Recently, H2 viruses have been identified widely in wild migratory birds worldwide [3–5], with H2N2, H2N3, and H2N7 viruses isolated from domestic ducks and pigs [6–9]. Circulation of these viruses in domestic animals increases the risk of human exposure [10, 11]. Live poultry markets (LPMs) are a major source of AIV dissemination and sites for potential AIV reassortment and interspecies transfers. An H2N8 AIV [A/duck/Zhejiang/ 6D10/2013(H2N8) (ZJ-6D10)] was isolated from a domestic duck during AIV surveillance of poultry in LPMs in Zhejiang Province, Eastern China, in 2013. The objective of this study was to elucidate the genetic relationships between this H2N8 AIV isolated from Eastern China and from birds in Asia and to determine the pathogenicity of this isolate in animals. Cloacal swabs were collected from domestic ducks in LPMs in Zhejiang Province, Eastern China, and viruses were isolated by inoculation of embryonated chicken eggs as described previously [12]. After incubation at 37 °C for 72 h, the allantoic fluid was harvested and tested by HA assay. The subtypes of the virus isolates were determined by conventional HA inhibition and NA inhibition assays. Aliquots of virus allantoic fluid stock were stored at -80 °C before use. RNA was extracted from the allantoic



Virus Genes (2016) 52:863–866

Fig. 1 Phylogenetic trees based on the nucleotide sequences of HA (positions 1–1695) and NA (positions 1–1413) comparing the H2N8 AIV isolated in this study to reference AIV sequences published in GenBank. The tree was created using the maximum likelihood

method and bootstrapped with 1000 replicates by the MEGA software version 6.0. The H2N8 virus characterized in this study is indicated by a triangle. The scale bar represents the unit of distance between sequence pairs

fluid using TRIzol, and reverse transcription was performed using the Uni12 primer: 50 -AGCAAAAGCAGG-30 . RT-PCR was performed using a One-Step RNA PCR Kit (TaKaRa, China). All of the viral gene segments were amplified with segment-specific primers and sequenced as described previously [13]. The nucleotide sequences were deposited into GenBank (accession numbers KX39437380) and analyzed using the BioEdit version DNA analysis software. Phylogenetic trees were constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0 [14].

Viral pathogenicity was evaluated in mice. Fifteen sixweek-old female BALB/c mice were inoculated intranasally with 106 egg infectious dose 50 (EID50) of virus in a 0.05 mL volume. Three mice were killed at each of the following time points: 3, 6, and 9 days post-inoculation (dpi). The lung, brain, heart, kidney, spleen, and liver were collected. The remaining six mice were observed to determine survival and weight loss for 14 dpi as described previously [15]. Embryonated chicken eggs were used to determine the EID50 using the method described by Reed and Muench [16].


Virus Genes (2016) 52:863–866


Table 1 Characteristics of the novel reassortant H2N8 avian influenza virus isolated from a domestic duck in Eastern China Virus

No. of survivors/no. tested

HI titer (log2)

Virus replication in experimentally infected mice Virus titers in organs of mice (log10 EID50/ml) Tissue



5.2 ± 0.8

3 days

6 days

9 days


3.0 ± 0

4.5 ± 0.7

3.0 ± 0





Heart Liver

0 2.0 ± 0

0 0

0 0









The 50 % egg infectious dose (EID50) was determined according to the recommendations of the Office International des Epizooties. Data represent mean ± SD. Sera were harvested at 14 days after infection from six mice which survived. Seroconversion was confirmed by the hemagglutination inhibition (HI) test

Phylogenetic analysis of the ZJ-6D10 gene sequences for polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic protein (PA), HA, nucleocapsid protein (NP), neuraminidase (NA), matrix protein (M), and nonstructural protein (NS) indicated that these genes were clustered in the Eurasian lineage. The HA phylogenetic tree showed that the ZJ-6D10 HA gene was derived from the H2 viruses responsible for the epidemic in Asian birds since 2010. The NA phylogenetic tree suggested that ZJ-6D10 has a high degree of nucleotide similarity with H3N8 viruses isolated in Eastern Asia between 2008 and 2014 and also indicated that ZJ-6D10 and the novel 2013 human H10N8 influenza virus have different ancestral N8 genes (Fig. 1). The remaining genes had a high degree of nucleotide similarity with different AIV subtypes (such as H3, H5, H7, and H10) isolated from aquatic birds and poultry throughout Eastern Asia, particularly domestic ducks from Eastern China (Table S1; Fig. S1). The ZJ-6D10 gene sequence similarity compared with its closest genetic relative is given in Table S1. The HA and NA genes had the highest similarity with A/duck/ Hokkaido/162/2013(H2N1) and A/duck/Zhejiang/4812/ 2013(H3N8), respectively. The PB2, PB1, PA, NP, M, and NS genes shared the highest similarity with H3, H7, and H10 isolates, which come from birds throughout Eastern Asia. Previous reports suggest that the HA genes of H2 AIVs (H2N2, H2N3, and H2N7) co-circulating in domestic ducks in Eastern and Southern China and the H2 AIVs underwent frequent reassortment with multiple virus subtypes [6, 7, 9]. Our findings suggest that ZJ-6D10 is a novel reassortant virus derived from multiple AIV subtypes isolated from aquatic birds and poultry in Eastern Asia. This indicates that a reassortment event between H2 and H3 viruses occurred in domestic ducks.

The HA cleavage site pattern (PQIESR/GL) identified from the deduced HA amino acid sequence indicates that ZJ-6D10 is a low-pathogenicity AIV. Amino acids at receptor-binding sites were highly conserved (Fig. S2). The Q226 and G228 (H3 numbering) receptor-binding sites in ZJ-6D10 are similar to those of the H2 AIVs in both the Eurasian and North American lineages. This suggests that ZJ-6D10 binds preferentially to alpha 2–3-linked sialic acid receptors that predominate among avian species [17, 18]. The L226 and S228 substitutions that are widely observed in human H2 viruses and increased affinity toward alpha 2–6-linked sialic acid receptors [17, 18] were not observed in ZJ-6D10. HA glycosylation is associated with virulence and virus affinity for the influenza virus receptor [19, 20]. Seven potential HA glycosylation sites at positions 25, 26, 38, 179, 300, 494, and 554 were detected in ZJ-6D10 (Fig S2). No substitutions associated with resistance to the NA inhibitor (His274Tyr) or amantadines (Ser31Asn) were observed in the NA and M2 proteins. Previous reports have shown that the Glu627Lys substitution in the PB2 protein is associated with AIV pathogenicity in mice [21, 22], and additional substitutions, such as Thr271Ala, Lys526Arg, Asp701Asn, and Ser714Arg, in the PB2 protein contribute to enhance viral polymerase activity and replication in mammalian host cells [22–24]. A previous study showed that the Thr97Ile substitution in the PA protein of H5 AIV also plays a key role in enhanced virulence in mice and is implicated in AIV adaptation to mammalian hosts [25]. However, none of these substitutions were observed in the corresponding ZJ-6D10 proteins. To evaluate the ability of ZJ-6D10 to replicate in mice, 15 mice were intranasally inoculated with 106 EID50 of ZJ6D10. ZJ-6D10 exhibited low pathogenicity in mice and was able to replicate without prior adaptation. At 6 dpi, higher ZJ-6D10 titers were detected in the lungs than at 3



dpi, and virus was detected in the liver of the mice; however, the virus was unable to replicate in the other organs. Survival rates of 100 % (6/6) up to 14 dpi were observed in infected mice (Table 1). Previous reports indicate the importance of domestic ducks to the genesis and evolution of AIV in China [26, 27]. Zhejiang Province is located in Eastern China and is an important location for migrating birds in East Asia. Birds from this region play an important role in AIV reassortment. A novel H7N9 virus associated with human deaths emerged in Eastern China in March 2013. This H7N9 virus is reported to be a reassortant of AIVs that have been co-circulating widely in birds, especially domestic ducks, in Eastern China [28, 29]. In this study, the H2N8 subtype AIV was first isolated in Eastern China, and this novel reassortant virus replicated in mice without prior adaptation. The isolation of this reassorted H2N8 virus from domestic ducks indicates the importance of this population in reassortment events leading to the emergence of new AIVs. Continued surveillance of domestic ducks in LPMs is required for early detection of AIV outbreaks in poultry and humans. Funding This study was funded by Grants from the National Science Foundation of the People’s Republic of China (81502852), Zhejiang Provincial Natural Science Foundation of China (Y15H190006), and the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Nos. 2015ZZ05 and 2016ZZ03).

Virus Genes (2016) 52:863–866

7. 8. 9. 10.


12. 13. 14. 15.

16. 17. 18.

19. 20. 21.

Compliance with ethical standards Conflict of interest The authors declare that they had no conflict of interest. Ethical approval The animal studies were approved by the First Affiliated Hospital, School of Medicine, Zhejiang University (No. 2015-015).


23. 24. 25.

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Wu H et al 2016

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