Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

Background Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus. Methodology and Principal findings We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. Conclusions These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.


Introduction
Human infection with a novel avian-origin influenza A (H7N9) virus, which is associated with severe respiratory symptoms and even deaths, was first reported in eastern China in April, 2013 [1,2]. There have been 135 diagnosed cases including 44 deaths as of Aug 14 th , attracting great attention worldwide [3].
The novel H7N9 virus is a triple reassortant virus, in which the HA and NA genes originated from A/duck/Zhejiang/12/2011 (H7N3) and A/wild bird/Korea/A14/2011 (H7N9) respectively, whereas the internal genes are closely related to A/brambling/ Beijing/16/2012-like viruses (H9N2), as previously described [1]. Most of the current researches have focused on the HA and NA genes since the Q226L mutation in the HA protein has been considered to change the binding capacity from avian species to human, and thus might increase the transmission ability in air [4][5][6]. However, various studies have also shown that the continuous reassortments occurred on the internal genes of avian influenza A virus played a key role in the direct interspecies transmission and triggering human infection [7,8]. This study, therefore, paid close attention to the origin and characteristics of the internal genes of the novel H7N9 virus. Different subtypes of avian influenza viruses (AIVs) possess different virulence and infectivity. Except for domestic poultry and wild birds, some AIVs including subtype H5, H7 and H9 had been detected from humans [9]. The high pathogenic avian influenza (HPAI) H5N1 viruses can spread rapidly in and between poultry, resulting in hundreds of millions of domestic birds affected and killed [10]. Only in China, there are approximately 100,000 domestic birds infected with the H5N1AIVs every year, causing huge economic losses [11]. The HPAI H5N1 viruses can also be widely transmitted by poultry products, poultry movements and migration of wild birds. In total of 63 countries had reported to detect the HPAI H5N1 viruses from poultry or wild birds [12]. In addition, the HPAI H5N1 virus is a great threat to human as 637 human beings had been infected since 2003 [13].
In contrast to H5N1, subtype H9 AIVs were generally considered to be low pathogenicity viruses causing mild disease among domestic poultry and wild birds [14][15][16]. Human infections with H9N2 AIVs have occasionally been reported in southern China and Hong Kong, but the clinical symptoms of the patients were mild to moderate and no deaths have occurred [17][18][19]. Even low pathogenic, H9N2 AIVs, however, possess high infectivity in both poultry and human. Since the first subtype H9N2 AIV was isolated in 1966 [20], the H9N2 AIVs have been monitored from multiple avian species in various regions [21][22][23][24][25][26]. The previous serological surveys have also pointed out that the positive rates for anti-H9N2 antibody were high in both poultry and human beings. In Iran, 23% to 87% of poultry-related workers possessed antibody for H9 [9]. Chinese studies also reported that 12.8% of chickens and 5.1% of poultry-related workers in Guangzhou area were seropositive for H9N2 [27]. Even in healthy individual, the prevalence of anti-H9N2 antibodies was reported to be around 2% [19,28].
Infections with earlier H7N9 AIVs were rarely reported in China or sporadically happened in a couple of countries [29,30]. No H7N9 virus has been monitored and isolated from poultry in Zhejiang province before 2013, which indicated the low transmissibility of the earlier H7N9 AIVs. However, the novel H7N9 viruses represented high transmissibility as the HPAI H5N1 and the H9N2 AIVs possessed. Our concern is whether we can find any common features on the basis of the genome sequences among the HPAI H5N1 viruses, the H9N2 and earlier H7N9 AIVs, as well as the novel H7N9 viruses, which could be the possible reason for the high infectivity of the novel H7N9 virus.
In this study, we monitored the environmental specimens collected from live poultry markets and isolated five avian H9N2 viruses from both H7 and H9 positive specimens. Meanwhile, the origin of the internal genes of the novel H7N9 virus was systematically illustrated by analyzing the entire sequences of 221 influenza viruses using RDP3 software. Phylogeny analysis and homological comparison were performed to determine the reassortment events. In addition, the specific amino acids possibly related to the high infectivity of the novel H7N9 viruses were analyzed by comparing the genome sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 AIVs.

Surveillance of AIVs from the environmental specimens
After the outbreaks of human infection with the novel H7N9 virus in Zhejiang province, China in 2013, we tested the environmental specimens collected from live poultry markets using real-time RT-PCR. Within the 82 environmental specimens collected from six cities, 33 (40.24%) were found to be positive for H7, 15 (18.29%) positive for H5 and 39 (47.56%) positive for H9. There were 23 (28.05%) found to be positive for both H7 and H9. Of the specimens positive for both H7 and H9, five AIVs were isolated, including A/environment/ Zhejiang/09/2013 (ZJ09), A/environment/Zhejiang/13/2013 (ZJ13), A/environment/Zhejiang/14/2013 (ZJ14), A/ environment/Zhejiang/15/2013 (ZJ15) and A/environment/ Zhejiang/16/2013 (ZJ16), in which two specimens were from Hangzhou city and three from Huzhou city. Ct values of the clinical specimens of the five isolates ranged from 10 to 14 for H9, and 17 to 35 for H7. After the virus isolates were obtained from allantoic liquids, the whole genome of the isolates were sequenced and aligned in Blast of NCBI. The results showed that the sequences of each gene shared the highest similarities with H9N2 AIVs strains, and thus the five Zhejiang isolates were identified as avian influenza A (H9N2) viruses. The details of five Zhejiang H9N2 AIVs are shown in Table 1.
A retrospective analysis on surveillance data for the AIVs in Zhejiang province between 2011 and 2012 was performed as well. In total 784 environmental specimens were tested by realtime RT-PCR assay to detect H5, H7 and H9 AIVs in 2011, in

Origin of the internal genes of the novel H7N9 virus
The evolutionary process of the novel H7N9 virus analyzed by RDP3 software showed that the six internal genes of the novel H7N9 viruses shared the highest similarities with ZJ16 (H9N2)-like viruses ( Figure 1). Significant reassortment signals were found on the internal genes of ZJ16 (H9N2)-like viruses, with acquiring NS gene from a H9N2 virus isolated from chicken in Jiangsu province in 2011, named A/chicken/ Dawang/1/2011 (H9N2), and other five genes from A/ brambling/Beijing/16/2012 (BJ16) (H9N2)-like strains.
Phylogenetic trees drawn on the basis of the amino acid sequences were used to confirm the reassortment events. The PB2 gene phylogenetic tree (Figure 2A) showed that five novel H7N9 strains were closest to Zhejiang H9N2 strains ZJ09,

Amino acids associated with the high infectivity of the novel H7N9 virus
In this study, the specific amino acids possibly related to the high infectivity of the novel H7N9 viruses were analyzed based on the criteria illustrated in Figure 3. As the novel H7N9 viruses, the HPAI H5N1 and the H9N2 AIVs were all defined as highly transmissible viruses, the amino acids which were identical amongst these three groups were selected first. Since the earlier H7N9 AIVs were considered as low infectivity, the identical amino acids between the earlier H7N9 viruses and the selected amino acid sites mentioned-above were excluded. The results are shown in Table 3.
In accordance with Figure 3 and the results in Table 3, a total of nine amino acids within the internal genes, including one in PB1 (aa113), three in NP (aa77, aa105 and aa377), three in M1 (aa15, aa101 and aa166), one in M2 (aa28), and one in NS1 (aa207) were found to be identical between the novel H7N9, the HPAI H5N1 and the H9N2 AIVs groups, but completely different with the earlier avian H7N9 group. Importantly, no amino acid on the HA and NA genes satisfied this requirement, therefore, only the nine specific amino acids showed a relationship with the high infectivity of the novel H7N9 virus.

Discussion
Since March 2013, a severe and fatal respiratory disease, characterized by high fever and severe low respiratory symptoms, has occurred continuously in China, mainly in Zhejiang and Jiangsu province, as well as Shanghai city [1,2,6]. The Chinese national influenza centre (CNIC) identified the pathogen as a triple reassortant avian-origin influenza A (H7N9) virus by testing the clinical specimens of the first three patients; meanwhile, the genetic origins of the viruses were also analyzed [1].
The novel H7N9 virus has infected 46 individuals resulting in eight deaths within one month in Zhejiang province, China. Except for the laboratory diagnosis of the cases, the environmental specimens collected from live poultry markets were monitored for H5, H7 and H9 AIVs in order to assess whether the novel H7N9 virus has infected the domestic poultry and circulated in the external environment. Interestingly, 23 specimens were detected to be positive for both H7 and H9 during this surveillance, however, the Ct value of H9 (Ct value 10-14) obtained from real-time RT-PCR assay were significant lower than that of H7 (Ct value [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and all the isolates obtained from the double-positive specimens were identified as H9N2 AIVs, which indicated the co-infection of H7 and H9 has existed in Zhejiang province, and that domestic poultry, especially chicken, has a close relationship with the reassortment between the novel H7N9 and avian H9N2 viruses. Based on the previous study, A/brambling/Beijing/16/2012 (BJ16), a H9N2 strain isolated from brambling in Beijing in 2012, were identified as the donor of the internal genes of the novel H7N9 virus [1]. However, A/environmental/Zhejiang/ 16/2013 (H9N2)-like strains showed the higher similarities with the novel H7N9 virus than BJ16. The surveillance on the environmental specimens revealed that A/environmental/ Zhejiang/16/2013 (H9N2)-like strains existed and circulated in the domestic poultry in Zhejiang province before the outbreak caused by the novel H7N9 virus. Since most of the human infections with the novel H7N9 virus occurred in the Yangtze River Delta, from an area point of view, it seems more reasonable that the six internal genes of the novel H7N9 virus originated from the viruses reassorted and circulated in the Yangtze River Delta. Unfortunately, there is no information about the avian H9N2 viruses isolated in Jiangsu, Zhejiang province and Shanghai city in 2012, it is therefore difficult to clearly know when the ZJ16 (H9N2)-like viruses emerged, how long they have circulated and who were their original host.
Human infection with the HPAI H5N1 virus was first reported in Hong Kong in 1997, resulting in 18 cases with six deaths from May 1997 to February 1998 [31]. The pathogen caused this outbreaks was identified as A/Hong Kong/156/97 (H5N1) strain. Genetic analysis on the whole genome sequence showed that the internal genes of A/Hong Kong/156/97 (H5N1) shared the highest homologies with H9N2 AIVs [31,32]. As same as the A/Hong Kong/156/97 (H5N1) strain, the six internal genes of the novel H7N9 virus were also originated from the H9N2 AIVs. The notable feature of both A/Hong Kong/ 156/97 (H5N1) and the novel H7N9 viruses is the increase of the infectivity after genetic reassortment, with a large amount of poultry and even human infected. Thus, the internal genes reassorted from H9N2 AIVs may affect the infectivity of the novel H7N9 viruses.
In this study, several subtypes of AIVs, including HPAI H5N1, avian H9N2 and earlier H7N9 viruses, were included to analyze the possible reason for the high infectivity of the novel H7N9 virus. These three subtypes of AIVs were all of avian origin and have been identified to cause human infections. The differences and similarities of amino acid sequences among the three subtypes of AVIs may result in different infectivity to humans. In this study, the HPAI H5N1, the H9N2 AIVs and the novel H7N9 viruses are characterized as high infectivity, therefore the identical amino acids shared amongst these three groups were selected as the high infectivity related sites, as shown in Figure 3. Since the earlier H7N9 AIVs are acknowledged low transmissibility [29,30], we removed the amino acids that the earlier H7N9 virus possessed from the selected sites. Interestingly, all the nine amino acids obtained, possibly associated with the high infectivity of the novel H7N9 virus, were located on the internal proteins and none were found on the surface proteins. It can be inferred that the internal genes, reassorted from avian H9N2 virus, may increase the infectious ability of the novel H7N9 virus.
Even though the novel H7N9 virus is highly infective and pathogenic to humans, it is generally low pathogenic to poultry [1,29]. Therefore, it is difficult to eliminate the novel H7N9 virus from the environment, the domestic poultry as well as the wild birds. Although the infection can be controlled in the same manner as the HPAI H5N1 viruses through slaughter of the poultry [31,33,34], widely existing avian H9N2 viruses will provide rich sources of the internal genes to H5, H7 and other subtype viruses [14,19,31]. Once the reassortment happened, the newly reassortant avian influenza viruses have the potential to cross the species barrier to infect humans. Therefore, the surveillance on mutations, evolutionary and reassortment of the H9N2 AIVs should be strengthened for the novel H7N9 viruses to be controlled.

Ethics Statement
Ethics committee of Zhejiang provincial Center for Disease Control and Prevention (CDC) approved this study. In order to control the outbreak of infection with the novel H7N9 virus, staff in municipal CDC took responsibility for disinfection of live poultry markets. To assess the disinfectant effect, the environmental specimens were collected before and after the disinfection. All the specimens used in this study were collected from environment before disinfection.

Specimens and virus isolation
In total 82 environmental specimens were screened for subtype H5, H7 and H9 AIVs using one-step real-time reversetranscriptase polymerase chain reaction (rRT-PCR) assays. The primer pairs and probes of the real-time RT-PCR assays were designed and provided by the Chinese national influenza centre (CNIC). The environmental specimens, including swabs of chicken cage, chicken manure and sewage of slaughter poultry, were collected from the live poultry markets in Zhejiang province, China during 1 st April to 2 nd May in 2013. Those specimens found to be positive for both H7 and H9 AIVs were inoculated to the allantoic sac of 9-to-11-day-old specific pathogen-free (SPF) embryonated chicken eggs for 48 hours at 35°C to propagate the virus.

RNA extraction and sequencing
RNA was extracted from the harvested allantoic liquids using the QIAamp Viral RNA Mini Kit (Qiagen). The whole genomes of five avian H9N2 viruses were performed by second generation sequencing in Applied Biosystems (ABI). The obtained sequences of each gene were aligned in BLAST of NCBI. The complete sequences of five Zhejiang isolates were submitted to GenBank by Zhejiang provincial centre for disease control and prevention (CDC).
A multiple sequence alignment was performed by the ClustalW program and the amino acid mutations were analyzed with the Highlight model using MEGA software (Version 5.0). Phylogenetic trees were drawn with the Neighbor-joining method of the MEGA software (Version 5.0) by means of bootstrap analysis with 1000 replications. Reassortment events on the six internal genes of the novel H7N9 viruses were searched by the RDP, GENECONV and MaxChi suites within the Recombination Detection Program (RDP3) software, according to the instruction and previous studies [36]. The highest P value was set as 0.01 in order to enhance the credibility of the results. Other parameters were used with default settings.

Phylogeny analysis
Phylogenetic trees were generated based on the amino acid sequences of 39 influenza viruses by means of the Neighborjoining (NJ) method of the MEGA software (Version 5.0), with the bootstrap analysis of 1000 replications.

Comparison on the amino acid sequences of three subtypes of AIVs
The multiple sequence alignments based on the amino acid sequences of the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 AIVs, as well as the novel H7N9 viruses were performed by using BioEdit software. Except for the novel H7N9 viruses, all the amino acid sequences were downloaded from GenBank. The GenBank accession numbers of the AIVs used for the comparison were shown in Table S1.
The comparative method and selection principle of the specific amino acids associated with high infectivity of the novel H7N9 virus is illustrated in Figure 3. The amino acids sequences on the whole genome of the four group AIVs were compared. The yellow region, representing high infectivity, was obtained by selecting the amino acids that were identical amongst the novel H7N9, the HPAI H5N1 and H9N2 AIVs, but different from the earlier H7N9 AIVs. Table S1. Genbank accession numbers of amino acid sequences for the AIVs used in this study. (DOC)