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Persistent detection of avian influenza A/H7N9 virus among poultry in Huzhou City, China, in the summer of 2013

Open AccessPublished:July 10, 2014DOI:https://doi.org/10.1016/j.ijid.2014.01.020

      Summary

      In eastern China, live poultry markets were successively re-opened in the summer of 2013 following their closure in April 2013. We detected influenza A/H7N9 RNA with positive rates from 4% to 22.2% among poultry samples in targeted markets in Huzhou City, China, from August 6 to September 24, 2013. Phylogenetic analyses of hemagglutinin and neuraminidase genes confirmed that the strain prevalent among poultry in Huzhou City in the summer of 2013 belonged to the same genotype as those capable of infecting humans. These results raise concern for a further outbreak of H7N9 in the cooler season.

      Keywords

      1. Introduction

      In February 2013, a new avian influenza virus, influenza A/H7N9, led to an outbreak in China, particularly affecting the Yangtze River Delta region.
      • Gao R.
      • Cao B.
      • Hu Y.
      • Feng Z.
      • Wang D.
      • Hu W.
      • et al.
      Human infection with a novel avian-origin influenza A (H7N9) virus.
      This virus had caused 136 confirmed cases and 45 deaths as of October 31, 2013 (http://www.nhfpc.gov.cn/yjb/s3578/201311/fb3e66ae0f5c4601a2ca137bd2625fd4.shtml, accessed November 13, 2013). The H7N9 virus was demonstrated to have originated from the reassortment or three earlier avian influenza viruses, H9N2, H7N3, and H7N9.
      • Gao R.
      • Cao B.
      • Hu Y.
      • Feng Z.
      • Wang D.
      • Hu W.
      • et al.
      Human infection with a novel avian-origin influenza A (H7N9) virus.
      • Liu D.
      • Shi W.
      • Shi Y.
      • Wang D.
      • Xiao H.
      • Li W.
      • et al.
      Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses.
      The majority of H7N9-infected individuals had a history of exposure to poultry,
      • Li Q.
      • Zhou L.
      • Zhou M.
      • Chen Z.
      • Li F.
      • Wu H.
      • et al.
      Preliminary report: Epidemiology of the avian influenza A (H7N9) outbreak in China.
      • Han J.
      • Jin M.
      • Zhang P.
      • Liu J.
      • Wang L.
      • Wen D.
      • et al.
      Epidemiological link between exposure to poultry and all influenza A(H7N9) confirmed cases in Huzhou City, China, March to May 2013.
      and the H7N9 virus was detected in live poultry markets, but not in large-scale poultry farms.
      • Shi J.Z.
      • Deng G.H.
      • Liu P.H.
      • Zhou J.P.
      • Guan L.Z.
      • Li W.H.
      • et al.
      Isolation and characterization of H7N9 viruses from live poultry markets—implication of the source of current H7N9 infection in humans.
      This indicated that poultry was the direct animal reservoir and that the live poultry markets, rather than the poultry farms, were the source of the H7N9 virus. Furthermore, unlike human infection, the infection of poultry with H7N9 virus did not result in the development of obvious disease. This increases the difficulty of routine surveillance of H7N9 virus among poultry. In order to effectively prevent human infections, the majority of live poultry markets were closed successively in eastern China starting April 6, 2013 and this closure effectively stopped new human infection events.
      • Xu J.
      • Lu S.
      • Wang H.
      • Chen C.
      Reducing exposure to avian influenza H7N9.
      Huzhou is a city in the north of Zhejiang Province and is located at the geographic center of the Yangtze River Delta (Figure 1A ). Twelve confirmed H7N9 cases were reported in Huzhou City.
      • Han J.
      • Jin M.
      • Zhang P.
      • Liu J.
      • Wang L.
      • Wen D.
      • et al.
      Epidemiological link between exposure to poultry and all influenza A(H7N9) confirmed cases in Huzhou City, China, March to May 2013.
      During the period April 4 to May 2, 2013, we detected H7N9 viral RNA among 64 of 405 poultry samples from some of the live poultry markets in Huzhou using the real-time RT-PCR protocol recommended by the World Health Organization (WHO; available at: http://www.who.int/influenza/gisrs_laboratory/a_h7n9/en/). This led to the subsequent closure of most live poultry markets in Huzhou City.
      • Han J.
      • Jin M.
      • Zhang P.
      • Liu J.
      • Wang L.
      • Wen D.
      • et al.
      Epidemiological link between exposure to poultry and all influenza A(H7N9) confirmed cases in Huzhou City, China, March to May 2013.
      On June 19, 2013, we collected samples from the scattered poultry-raising households in a village as part of seasonal influenza surveillance and did not detect H7N9 RNA among the poultry.
      Figure thumbnail gr1
      Figure 1Testing for H7N9 virus and average daily temperature and relative humidity in Huzhou City, from April 1 to September 31, 2013. (A) Geographic location of Huzhou City. (B) Surveillance of H7N9 and the average daily temperature and relative humidity from April 1 to September 31, 2013. The black and pink triangles indicate H7N9 RNA being detected and not being detected, respectively. The dates of H7N9 surveillance and the numbers of H7N9-positive and total poultry samples are shown below the triangles. (C) Numbers of poultry markets selected for H7N9 surveillance in Huzhou City, China, from August to September 2013. The information on markets selected for H7N9 surveillance from April to May 2013 were reported previously by Han et al.
      • Han J.
      • Jin M.
      • Zhang P.
      • Liu J.
      • Wang L.
      • Wen D.
      • et al.
      Epidemiological link between exposure to poultry and all influenza A(H7N9) confirmed cases in Huzhou City, China, March to May 2013.
      On June 19, only several scattered poultry-raising households were covered. *A total of 13 markets were monitored, including one market that was monitored three times and one that was monitored two times.
      High temperature and high relative humidity are unfavorable for the transmission of influenza virus.
      • Lowen A.C.
      • Mubareka S.
      • Steel J.
      • Palese P.
      Influenza virus transmission is dependent on relative humidity and temperature.
      In particular, a temperature over 30 °C or a relative humidity over 80% can completely block transmission. This implies that an epidemic of influenza will decrease or even disappear during the summer. The summer of 2013 was the hottest summer in eastern China on record and it was believed to be unfavorable for the survival and transmission of the H7N9 virus. Therefore, the local government readily relaxed vigilance for H7N9 virus and re-opened the live poultry markets during the hot weather. The continuing hot weather in Huzhou City (Figure 1B), together with the demands of the poultry industry, led to the successive re-opening of the live poultry markets in Huzhou City starting on August 2–3, 2013.2.

      2. Methods

      In response to the re-opening of the live poultry markets, we performed a series of H7N9 surveillance investigations in live poultry markets on August 6 and 20, and September 16 and 24, 2013. A total of 13 live poultry markets were investigated and 113 poultry samples, including 92 poultry feces samples, 20 swab samples from culling benches, and 1 sewage sample, were collected during this period. Among these markets, the agricultural and sideline product market of northern Zhejiang and the Lianhua Zhuang farmer's market were monitored three (August 6 and 20, and September 16) and two (August 6 and 20) times, respectively; the others were investigated only once (Figure 1C).

      3. Results

      As for our previous studies,
      • Han J.
      • Jin M.
      • Zhang P.
      • Liu J.
      • Wang L.
      • Wen D.
      • et al.
      Epidemiological link between exposure to poultry and all influenza A(H7N9) confirmed cases in Huzhou City, China, March to May 2013.
      • Han J.
      • Niu F.
      • Jin M.
      • Wang L.
      • Liu J.
      • Zhang P.
      • et al.
      Clinical presentation and sequence analyses of HA and NA antigens of the novel H7N9 viruses.
      the WHO-recommended protocol was again used for the detection of H7N9 viral RNA. Among the samples obtained, 17 (15.0%) were positive for H7N9 viral RNA (Figure 1B). Although the maximum daily temperatures on those days reached 31.6–39.7 °C, H7N9 RNA was persistently detected in the markets targeted from August 6 to September 24, 2013, with positive rates from 4% to 22.2% (Figure 1B). The positive samples were found in four of 13 (30.8%) targeted markets (Figure 1C). In particular, the agricultural and sideline product market of northern Zhejiang was found to be positive for H7N9 viral RNA at all three surveillance times from August 6 to September 16, 2013, implying that this market might be one of the most important sources of the H7N9 virus.
      To confirm that these viruses were similar to the new H7N9 viruses infecting humans, hemagglutinin (HA) and neuraminidase (NA) genomic fragments were amplified from the H7N9 RNA-positive poultry samples using universal primer sets, as described previously,
      • Hoffmann E.
      • Stech J.
      • Guan Y.
      • Webster R.G.
      • Perez D.R.
      Universal primer set for the full-length amplification of all influenza A viruses.
      and were sent for sequencing. The sequences obtained in this study are available on request from the authors. Phylogenetic analyses of the HA and NA fragments showed that the new H7N9 strains formed a well-supported clade (with a bootstrap value of 100%) (Figure 2). The sequence from a poultry sample collected on August 6, 2013 in Huzhou City, is included in the new H7N9 clade, and clusters closely with other H7N9 strains, including the strain from the first patient in Huzhou City (A/Huzhou/1/2013)
      • Han J.
      • Niu F.
      • Jin M.
      • Wang L.
      • Liu J.
      • Zhang P.
      • et al.
      Clinical presentation and sequence analyses of HA and NA antigens of the novel H7N9 viruses.
      (Figure 2). This suggests that the strains prevalent among poultry in the summer of 2013 in Huzhou City are the same genotype as those capable of infecting humans.
      Figure thumbnail gr2
      Figure 2Phylogenetic trees of influenza hemagglutinin (HA) and neuraminidase (NA) sequences. The trees were constructed using the neighbor-joining method implemented in MEGA 5.0, and bootstrap analysis with 1000 replications was performed to assess the reliability of the branching pattern. The sequences from poultry and the first patient in Huzhou City are highlighted with red solid and open circles, respectively.

      4. Discussion

      The detection of the H7N9 virus during hot weather not only indicates the persistent prevalence of H7N9 virus among poultry in Huzhou City, but also suggests that the new H7N9 virus has a better fitness or adaptation to poultry even in hot weather conditions. From October 2013 to January 20, 2014, four H7N9 cases were newly confirmed in Huzhou City. As at January 13, 2014, the national H7N9 case number had increased to 170, and most of the new cases in the 2013 winter were located in the eastern China region. This is particularly alarming since both the winter and the spring seasons will be more favorable for influenza virus transmission than the other seasons. The continuous detection of H7N9 virus in the agricultural and sideline product market of northern Zhejiang in Huzhou City in the summer of 2013 implies that it is an important transmission source of the H7N9 virus in this region. Distinct from the large-scale poultry farms where no H7N9 virus was detected, the vast majority of the live poultry markets in China have mixed multiple species of poultry kept in a narrow space. The mixing of multiple species of poultry in a narrow space forms a dynamic environment facilitating viral interspecies transmission. Therefore, we must strengthen routine surveillance of live poultry markets to effectively prevent a large-scale outbreak of the H7N9 virus in the winter of 2013 and the coming spring.

      Acknowledgements

      We thank Meihua Jin, Xiaofang Wu, Deshun Xu, Lei Ji, Liping Chen, and Xiaoqi Liu for their technical assistance. This work was supported by grants from the China National Mega-projects for Infectious Diseases (2012ZX10004211-002 and 2013ZX10004101-005) and the Public Health Key Disciplines in Shanghai-Health Microbiology (12GWZX0801).
      Conflict of interest: No conflict of interest to declare.

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