Cocirculation of Three Hemagglutinin and Two ...

Cocirculation of Three Hemagglutinin and Two Neuraminidase Subtypes of Avian Influenza Viruses in Huzhou, China, April 2013: Implication for the Origin of the Novel H7N9 Virus Jiankang Han,a Lili Wang,b Jia Liu,b Meihua Jin,a Fangyuan Hao,b Peng Zhang,a Zhao Zhang,b Dong Wen,a Xiaofang Wu,a Guangtao Liu,a Lei Ji,a Deshun Xu,a Dongming Zhou,b Qibin Leng,b Ke Lan,b Chiyu Zhangb Huzhou Center for Disease Control and Prevention, Huzhou, Zhejiang, Chinaa; Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, Chinab

A

novel H7N9 avian influenza virus (AIV) has led to an outbreak of human infection in China (1–3). The Yangtze River Delta Region, including the Shanghai, Jiangsu, Zhejiang, and Anhui Provinces, is the worst-hit area of the H7N9 outbreak (2). As of 20 May 2013, 33, 27, 46, and 4 patients were confirmed to be infected by the novel H7N9 virus in the Shanghai, Jiangsu, Zhejiang, and Anhui Provinces, respectively. The H7N9 virus was demonstrated to originate via reassortment events between three earlier AIVs, H7N3, H7N9, and H9N2 (1, 4). Both H7N3 and H9N2 were two circulating influenza virus lineages in eastern China, and the earlier H7N9 lineage may have been introduced into wild-bird habitats in eastern China by migratory birds. H7N9 virus was detected in some live-poultry markets in affected areas, indicating that poultry was the direct animal reservoir. The majority of poultry in markets was from farms, and a few birds were from individual farmers. H7N9 RNA was not detected in large-scale poultry farms (5), indicating that farms were not the source of H7N9 virus and suggesting that the freeranging domestic fowl raised by some individual farmers might be associated with the origin of the H7N9 virus because they are accessible to wild ducks or wild birds. However, where the H7N9 virus originated still remains a mystery (3). We previously reported 12 confirmed A(H7N9) cases in Huzhou City, a city in the northern portion of Zhejiang Province (Fig. 1) (6, 7), accounting for about 9.0% (12/134) of all cases in China as of 20 May 2013. We collected poultry feces, waste (swab samples from culling benches), and sewage from the live-poultry markets in Huzhou City during April 2013 and detected influenza A(H7N9) viral RNA among these markets, as well as a local farmer’s courtyard, using real-time reverse transcription-PCR (RTPCR) (6). An epidemiological link between poultry exposure and H7N9 virus infection in Huzhou City was demonstrated (6). To trace the origin of the H7N9 virus, we further amplified and sequenced the H7N9 sequences from confirmed cases and poultryrelated samples from Huzhou City. In order to obtain complete information on the influenza pandemic in Huzhou City, we used TA clones with the pMD18-T vector (TaKaRa) for cloning hemagglutinin (HA) and neuraminidase (NA) fragments of all samples. At least 30 colonies from each fragment were subjected to sequencing. The obtained sequences were subjected to subtype and phylo-

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genetic analyses. The sequences with subtype designations have been submitted to the Global Initiative on Sharing Avian Influenza Data (GISAID) EpiFlu Database under the accession numbers EPI464516 to EPI464579, and the other sequences can be obtained upon request from the authors. Genotyping analysis showed that cloned sequences from the same sample, except HA fragments of two sewage samples (C58 and C291) and two waste samples (C56 and C57), had identical subtype results (Table 1). For C56, C57, and C58, both the H7 and H9 subtypes were detected, and for C291, the H7, H9, and H5 subtypes were detected (Table 1 and Fig. 2A). Because sewage and chicken waste represents a mixture of samples from multiple chickens, the detection of H7, H9, and H5 among these samples suggests a cocirculation of three HA subtypes among poultry in Huzhou City. In addition to this, we detected the H9 subtype among four other poultryrelated samples (C73, C143, C169, and C181) (Table 1 and Fig. 2A), supporting cocirculation. Similarly, for NA genomic fragments, besides N9, we also detected N2 subtypes in four poultry samples (Table 1 and Fig. 2B), indicating the cocirculation of the N9 and N2 subtypes. In particular, among five samples, C72, C73, C75, C76, and C77, which were collected from one retail livepoultry stall, C72, C73, and C77 belong to the N9 subtype, while C75 and C76 belong to the N2 subtype. Accordingly, C72 belongs to the H7 subtype, while C73 belongs to the H9 subtype (Table 1). These findings suggest that different AIV subtypes (at least H7N9 and H9N2) were cocirculating among poultry in Huzhou City, as observed in other cities (8, 9). It is worth noting that no influenza subtypes other than the novel H7N9 subtypes were found among patients’ samples. Interestingly, we found potential H9N9 reassortants among

Received 11 November 2013 Accepted 6 March 2014 Published ahead of print 12 March 2014 Editor: A. García-Sastre Address correspondence to Chiyu Zhang, [email protected], or Ke Lan, [email protected]. J.H., L.W., J.L., and M.J. contributed equally to this work. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03319-13

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We detected three avian influenza hemagglutinin (HA) subtypes (H7, H9, and H5) and two neuraminidase (NA) subtypes (N9 and N2), as well as H7N9-related H9N9 reassortant intermediates, cocirculating among poultry in Huzhou, China, during April 2013. The results of our study reveal not only that Huzhou is one of the geographic origins of the novel H7N9 virus but also that cocirculation poses a potential threat to humans in the future.

Cocirculation of Various Avian Inﬂuenza Viruses

tions of two natural wild-bird habitats in Huzhou City.

two poultry-related samples. They are A/chicken/Huzhou/C73/ 2013 and A/sewage/Huzhou/C169/2013 (Table 1). The HA genes of the two H9N9 strains closely clustered with the H9N2 strains circulating in Huzhou City, and the NA genes clustered within the novel H7N9 clade, which closely clustered with the earlier H7N9 strains circulating in Korea (Fig. 2A and B). These findings suggest that the potential H9N9 strains originated via the reassortment between H9N2 and the earlier H7N9 viruses and that they are

TABLE 1 Summary of genotype results of the poultry-related samples from Huzhou City during April 2013a Subtype(s) of:

Sample code

Sample type

Strain designation

HA

NA

Internal gene(s) (cluster/group)

C54 C55

Feces Feces

A/chicken/Huzhou/C54/2013 A/chicken/Huzhou/C55/2013

H7 NotAv

NotAv N9

C56 C57 C58

Waste Waste Sewage

A/chicken waste/Huzhou/C56/2013 A/chicken waste/Huzhou/C57/2013 A/Sewage/Huzhou/C58/2013

H7, H9 H7, H9 H7, H9

NotAv N2 N9

C70 C72 C73 C75 C76 C77 C143

Feces Feces Feces Feces Feces Feces Throat swab

A/chicken/Huzhou/C70/2013 A/chicken/Huzhou/C72/2013 A/chicken/Huzhou/C73/2013 A/chicken/Huzhou/C75/2013 A/chicken/Huzhou/C76/2013 A/chicken/Huzhou/C77/2013 A/chicken/Huzhou/C143/2013

NotAv H7 H9 NotAv NotAv NotAv H9

N9 N9 N9 N2 N2 N9 N2

C169 C181

Sewage Feces

A/Sewage/Huzhou/C169/2013 A/chicken/Huzhou/C181/2013

H9 H9

N9 NotAv

C191

Waste

A/chicken waste/Huzhou/C191/2013

NotAv

N9

C291 C357

Sewage Sewage

A/sewage/Huzhou/C291/2013 A/sewage/Huzhou/C357/2013

H5, H7, H9 H7

N9 N9

M, NS (H7N9) NS (H7N9), M (immediately outside the H7N9 clade) M, NS (H7N9) M, NS (H7N9) NS, PB1, PB2 (H7N9), M, NP (immediately outside the H7N9 clade) M, NS (H7N9) M, NS, PB1, PB2 (H7N9) M, NS (H7N9) M (H9N2), NS (H7N9) M (H9N2), NS (H7N9) M (H9N2), NS (H7N9) M (H9N2), NS, PB1 (immediately outside the H7N9 clade), NP, PB2 (outside the H7N9 clade) M (outside the H7N9 clade) M, NS (H9N2), NP (immediately outside the H7N9 clade) M, NP (immediately outside the H7N9 clade), NS (H7N9) NP (H7N9) NotAv

a

NotAv, not available. The internal gene groups are defined based on the phylogenetic trees shown in Fig. 2C to H. Some internal gene sequences for certain samples are not available.


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FIG 1 Geographic location of Huzhou City. Two triangles indicate the loca-

closely related to the putative evolutionary intermediates of the novel H7N9 virus. Phylogenetic analyses of six other genomic sequences show that the majority of H7N9 strains from patients and poultry-related samples in Huzhou City closely clustered with the H7N9 strains from patients and environments in surrounding regions (Shanghai and Anhui), forming a well-supported clade of the novel H7N9 virus (with a bootstrap value of ⬎85, except in the M gene tree) (Fig. 2C to H). The two H9N9 reassortants were included in and/or were immediately outside the novel H7N9 clade (Fig. 2C and D), suggesting that they are closely related to the novel H7N9 virus in internal genes. In addition, we found that some sequences from poultry-related samples in Huzhou City have closer genetic relationships with the H7N9 virus in six internal genes than other earlier H9N2 strains circulating in the Yangtze River Delta Region since they were located immediately outside the novel H7N9 clade in the trees of six internal genomic sequences (especially the M and NP genes) (Fig. 2C to H). These findings indicate that they shared a most recent common ancestor (MRCA) with the novel H7N9 virus but were evolutionarily more ancient than the latter, at least in six internal genes, implying that the H7N9 virus may have originated from them. A good case in point is A/chicken/Huzhou/C143/2013(H9N2), which represents the strain most similar to the parental H9N2 strain involved in the generation of the novel H7N9 virus. We further inferred the time to the MRCA (tMRCA) of the novel H7N9 virus using the Bayesian Markov chain Monte Carlo (MCMC) analysis implemented in BEAST v1.6.1 (10). The analysis was performed using an uncorrelated lognormal relaxedclock model in an HKY plus I plus G4 nucleotide substitution

Downloaded from http://jvi.asm.org/ on October 20, 2015 by guest FIG 2 Phylogenetic analyses of eight genomic sequences of avian influenza viruses from poultry-related samples in Huzhou. (A to H) Phylogenetic trees of the HA, NA, M, NS, NP, PA, PB1, and PB2 genes, respectively. The phylogenetic trees were constructed with MEGA 5.0 using the neighbor-joining method. The reliability of topologies was estimated by performing bootstrap analysis with 1,000 replicates, and only bootstrap values of ⱖ80 were shown at the corresponding nodes. Because A/Shanghai/1/2013 was isolated from the first confirmed case in China and represented a relatively earlier form of the novel H7N9 virus, the novel H7N9 clade was identified according to the position of A/Shanghai/1/2013 in all trees except the NP tree, in which A/Shanghai/1/2013 clusters with an H9N2 strain isolated from a duck in Shanghai, far away from the other H7N9 strains. The position of A/Shanghai/1/2013 in the NP tree may imply that its NP gene originated from an additional reassortment with the H9N2 strain circulating in Shanghai. The cloned sequences from four samples that contain different HA subtypes are highlighted by their corresponding sample codes. A pink triangle represents avian influenza reassortant H9N9 lineages. The green arrows indicate the H9N2 strain [i.e., A/chicken/Huzhou/C143/2013(H9N2)] that is most closely (genetically) related to the novel H7N9 virus. If the cloned sequences from the same sample had identical subtype results, only one of them was used in the phylogenetic analyses.

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FIG 2 continued

model under a constant coalescent model. The MCMC analysis was run for 200 million generations until convergence with sampling every 10,000 generations. The effective sample size of ⬎200 for all parameters was checked using Tracer v1.5. We found that the NA gene of the novel H7N9 strains had an estimated tMRCA of 2.18 years (95% highest posterior density, 1.08 to 3.42 years ago), earlier than those of HA genes (1.53 years ago) and six internal genes (0.53 to 1.9 years ago) (Fig. 3A). These findings indicate that during the generation of the novel H7N9 virus, the N9 NA gene was first reassorted into the H9N2 genome and then the H7 HA gene was reassorted into the genome of the reassortant intermediate H9N9 virus (Fig. 3B).


The cocirculation of parental strains is a prerequisite for the generation of a novel influenza virus reassortant. Previous studies demonstrated that reassortment between three earlier H7N3, H7N9, and H9N2 lineages led to the generation of the novel H7N9 virus (1, 4) and that poultry was the native reservoir of these viruses (7, 11). Temporal dynamics analysis shows that the novel H7N9 virus has had a very short history since its generation (4), implying that the parental strains leading to the novel H7N9 virus, and even the evolutionary intermediates (i.e., H9N9) of the novel H7N9 virus, may still be cocirculating in areas where the virus originated. The temporal dynamics data from a previous study (4) and this

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study (Fig. 3A) suggest that the novel H7N9 virus originated from at least two rounds of reassortment events (Fig. 3B). The first round of reassortment led to the formation of the evolutionarily intermediate lineage H9N9 by replacing the N2 NA gene of the H9N2 virus with the N9 NA gene during November 2010. Then, the novel H7N9 virus originated via a second round of reassortment during July 2011, during which the H7 HA gene was reassorted into the genome of the H9N9 virus (Fig. 3B). The model implies that the strains similar to the parental H9N2 strains and the evolutionary intermediates (i.e., H9N9) might still be cocirculating in the geographic region of origin of the novel H7N9 virus. The NA gene of the novel H7N9 virus was derived from the earlier H7N9 lineage circulating among migratory birds along the East Asian flyway (1, 4), implying that in the Yangtze River Delta Region, the novel H7N9 virus most likely originated in regions having natural habitats for migratory birds. Huzhou City lies at the border among the Jiangsu, Zhejiang, and Anhui Provinces and is the geographic center of the Yangtze River Delta Region (Fig. 1). It faces the south side of Taihu Lake, the third biggest freshwater lake in China, and has two natural wetlands for over 160 kinds of wild birds, including migratory birds, thereby being a potential candidate for the region of origin of the H7N9 virus. We detected several H9N2 strains [e.g., A/chicken/Huzhou/C143/2013 (H9N2)] that were close genetically related to the novel H7N9 virus in six internal genes and the H7N9-related reassortant intermediates (i.e., H9N9 strains) among poultry-related samples in Huzhou City (Fig. 2), which strongly suggests that Huzhou is, at least, one of geographic regions of origin of the novel H7N9 virus. Summer is climatically unfavorable to the transmission of influenza virus (12). From spring to summer (April to September) of 2013, we continuously detected the H7N9 virus among poultryrelated samples in several agricultural and sideline-product markets in Huzhou City (6, 13), supporting the supposition that Huzhou is an important source of transmission of the H7N9 virus. On the other hand, we found that three avian influenza HA subtypes (H7, H9, and H5) and two NA subtypes (N9 and N2) were still cocirculating among poultry in Huzhou City (Fig. 2A and B), which provides an ideal dynamic environment for the ongoing generation of various H7N9-related reassortant lineages.

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Several avian influenza A viruses (e.g., H7N2, H7N3, H7N7, H5N1, etc.) led previously to sporadic human infections in some countries/regions (14, 15). This time, only the novel H7N9 virus acquired the ability to infect humans (16, 17). However, we do not exclude the possibility that H7N9-related reassortants (e.g., H9N9 viruses) will evolve the ability to infect humans in the future (18). Therefore, cocirculation of these HA and NA subtypes and the emergence of H7N9-related reassortants pose a potential threat to humans. There are large numbers of live-poultry markets in China. Although the 2013 spring outbreak of H7N9 influenza led to a shortterm (May to August 2013) closure of live-poultry markets in the Yangtze River Delta Region, most of these markets have now been reopened. Low temperatures and low relative humidity favor the transmission of influenza viruses (12, 19). After the winter of 2013 to 2014, a new wave of H7N9 outbreaks occurred in China. This wave of H7N9 epidemics is more severe than that of spring of 2013. From 1 January to 16 February 2014, the national number of new H7N9 infections had reached 204, far exceeding the number (134) of the spring of 2013. Furthermore, the new H7N9 virus has a better adaptation to poultry even under hot weather conditions (13). Therefore, to avoid potential crises in the future, constant surveillance of influenza subtypes in poultry in affected areas is urgently needed. Furthermore, multiple species of poultry are mixed in narrow spaces in most live-poultry markets in China. The mixing of multiple species of poultry forms a microenvironment for interspecies transmission and cocirculation of different AIVs. Standard production and supply chains for poultry also urgently need to be developed in China. ACKNOWLEDGMENTS This work was supported by grants from China National Mega-projects for Infectious Diseases (2012ZX10004211-002 and 2013ZX10004101005) to K.L. and the Li Ka-Shing Foundation to Q.L.

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FIG 3 Temporal dynamics (A) and model for reassortant origin (B) of the novel H7N9 virus.


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