Background
A particular subtype of influenza A virus, highly pathogenic avian influenza (HPAI) virus H5N1, is transmitted by contact with infected birds [
1]. It is epizootic in many bird populations, especially in Southeast Asia. Clade 2.2 of the virus has spread globally, including Europe, the Middle East and Africa after first appearing in Asia in 2005 [
2]. The spatial spread of H5N1 avian influenza and long-term persistence of the virus in some regions has had an enormous impact on the poultry industry and presents a serious threat to the health of humans and migratory birds [
3,
4]. It has been 17 years since the first case in geese of H5N1 avian influenza was discovered in Hong Kong in 1996 [
5]. As of 19 May 2013, H5N1 has caused 628 human cases of influenza in 15 different countries, with 374 deaths.
The high lethality and virulence of H5N1, its epizootic presence, its increasingly large host reservoir, its significant ongoing mutations, and its potential transmissibility between humans, make it one of the greatest current pandemic threats [
6]. Substantial progress has been made in researching various aspects of the virus and preparing for a potential influenza pandemic [
7,
8]. Several studies on the global spread of avian flu using phylogenetic relationships of virus isolates have indicated that migratory bird movements, and trade in poultry and wild birds could explain the pathway for introduction events into different countries [
9,
10]. However, the underlying mechanism of the long-term persistence of the virus and its various spatiotemporal transmission pathways, with their corresponding genetic footprints, remain poorly understood.
China is one of the world primary producers of poultry products [
11], and is among the regions most affected by H5N1 [
12]. Poultry production generates 16 million tons of meat and 27 million tons of eggs annually in China [
11]. In 2004, the total loss caused by HPAI virus H5N1 was 4.5 billion US dollars in China. To control H5N1 infection in poultry, many countries have implemented a vaccination policy, including China, Vietnam, Indonesia and Egypt. In Mainland China, a poultry vaccine was first used at the end of 2004. Over 55 billion doses of vaccines were applied to control the outbreaks between 2004 and 2008 [
13]. Antigenic variants of H5N1 avian influenza virus have occurred along with its spatiotemporal transmission. Furthermore, vaccination may change the evolutionary dynamics of H5N1 virus [
14,
15]. Vaccine strains can be selected from the seeding region of Southeast Asia where its genetic and antigenic characteristics can be determined earlier for human or avian influenza [
2,
16]. However, the effect of vaccination in China has rarely been studied and is discussed in the present study.
Here, we applied phylogenetic analysis, geospatial techniques, and time series models to investigate the spatiotemporal pattern of H5N1 outbreaks in China and the effect of vaccination on the dynamics of virus evolution. These data, combined with spatiotemporal information on the H5N1 outbreaks and viruses, were used to understand viral evolutionary dynamics from the beginning to its circulation in China. This study aimed to illustrate the spatiotemporal pattern of H5N1 outbreaks in China, understand the role of migratory birds and poultry in contributing such a pattern, and the effect of vaccination on the dynamics of virus evolution.
Discussion
In 1996, A/goose/Guangdong/1/1996 (H5N1), the precursor of currently circulating HPAI H5N1 virus was identified in China [
42]. The virus has circulated for 17 years, and presented an imminent threat to humans, poultry production, and wild animals in China [
43,
44]. Our study provides insight into the spatiotemporal pattern of H5N1 outbreaks in China and the dynamics of virus evolution. The results showed obvious spatiotemporal clusters of H5N1 outbreaks on different scales associated with two transmission modes of H5N1 viruses. Viral evolutionary dynamics were analyzed, and the effect of vaccination on virus circulation in China was identified.
Our time series analysis indicated a significant temporal relationship between poultry outbreaks, human cases, and wild-bird outbreaks. The current transmission chain of H7N9 virus in the country coincides with this H5N1 path. Results showed that human cases correlated with poultry outbreaks with a 1–4-month lag, which indicated that the infection may have started in poultry from live-bird markets and then transmitted to humans, even though the timing of wild-bird infection is still uncertain [
45,
46]. It is postulated that the virus might have undergone a period of time in poultry and the environment without being detected [
47,
48]. With an increase in imported poultry or active poultry production to meet the demand prior to Lunar New Year activities, most poultry outbreaks occur during these festivals in China [
49]. Human infections occurred 1–4 months thereafter because of dense human population, increased exposure, and possibly evolved high-affinity binding of the virus to human receptors. Wild-bird infections might occur last (correlated with human cases with a 1–3-month lag) after exposure and sufficient population of wild species. Many wild bird species winter in Southern China, and start spring migration in early April, and during these periods, wild birds may be infected directly through contact with infected poultry, or the environment [
50].
The transmission mode of poultry was faster, with a shorter cycle (Figure
2e). Additionally, the evolving capability for sustained transmission across species barriers represents a major adaptive challenge, because the number of required mutations is often large [
51]. However, a vast number of poultry hosts serve as a reservoir, providing a sufficient population basis for accumulating such mutations [
52]. Poultry may therefore play a crucial role in the avian influenza epidemics in the country.
Study of the spatiotemporal pattern of outbreaks is important in the prevention and control of epidemics over large regions [
53]. The seasonal characteristics of outbreaks in different hosts means that as the season alternates, epidemic areas shift significantly [
17]. The epidemic regions will naturally be affected by future climate change [
54,
55]. Poultry outbreaks concentrate in January, February, June and November. Wild-bird outbreaks concentrate in January and February in Hong Kong, and April and May in Northeastern China and Qinghai–Tibet Plateau. Through spatiotemporal pattern analysis, we can identify possible areas of subsequent outbreaks around epidemic areas. We can calculate an appropriate radius for prevention and culling, and establish early warning systems for regions potentially affected by outbreaks. The results of this study will help to develop appropriate prevention and control policies toward various host outbreaks in different time periods, to avoid the possibility of continuous large outbreaks.
A culling plus vaccination mixed strategy was initiated for control of HPAI H5N1 outbreaks in Mainland China [
56]. The actual effect of vaccination in different years and epidemic regions is debated. Although direct association between vaccination and H5N1 virus evolution is difficult to establish, we found that both evolutionary rates and positively selected sites were affected by vaccination for H5N1 in China. OIE has recognized that the emergence of clade 2.3.2.1 virus was one of the genetic mutations occurring as part of neutral virus evolution [
57]. However, we believe that the mutation possibly resulted from vaccination, and some mutation sites in clade 2.3.2.1 virus exhibited a strong signature of positive selection, which was against the signature of neutral evolution.
When an outbreak occurs in many regions, simple culling may not be effective. This is also true in some other infectious diseases [
12]. In developing countries, controlling virus spread by only long-term, large-scale culling is an immeasurable encumbrance on human living standards [
58]. Therefore, vaccination in combination with culling may be the most appropriate way for the country to control infections. The evolutionary patterns and spatiotemporal distribution of the virus are important in making targeted vaccination policy and developing appropriate prevention measures [
59]. An appropriate vaccination strategy that includes immunity planning directed at the spatiotemporal distribution of the circulating virus and its possible evolutionary pattern is more important than efficacy of the vaccine itself [
60].
The limitations of this study should be mentioned. First, the spatiotemporal information of H5N1 virus depended on sampling. Virus samples may have been concentrated in supposed high-risk areas, which could have led to data bias. Second, H5N1 outbreak information was collected from a passive surveillance system, and data quality varied significantly across provinces.
Funding sources
This research was supported by the Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407), and the National Natural Science Foundation of China (41271099). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The sources of funding for each author:
H.Y.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407), and the National Natural Science Foundation of China (41271099).
Y.C.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407).
L.D.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407).
S.Z.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407), and the National Natural Science Foundation of China (41271099).
X.L.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407), and the National Natural Science Foundation of China (41271099).
R.Y.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407).
B.X.: Ministry of Science and Technology, China, National Research Program (2010CB530300, 2012CB955501, 2012AA12A407), and the National Natural Science Foundation of China (41271099).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HT and BX designed the overall scope of the research. HT, YC, LD, SZ, XL and SH analyzed the data. XL, RY and BX contributed reagents/materials/analysis tools. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.