Avian-Origin Influenza A(H7N9) Infection in Influenza ...

BRIEF REPORT

Avian-Origin Influenza A(H7N9) Infection in Influenza A(H7N9)– Affected Areas of China: A Serological Study Shigui Yang,1,2,a Yu Chen,1,2,a Dawei Cui, Hangping Yao,1,2 Jianzhou Lou,1 Zhaoxia Huo,1 Guoliang Xie,1 Fei Yu,1 Shufa Zheng,1 Yida Yang,1 Yixin Zhu,1 Xiaoqing Lu,1 Xiaoli Liu,1 Siu-Ying Lau,2,3 Jasper Fuk-Woo Chan,3 Kelvin Kai-Wang To,3 Kwok-Yung Yuen,2,3 Honglin Chen,2,3 and Lanjuan Li1,2 1

Serological surveillance conducted in areas of an outbreak of influenza A(H7N9) infection in China found no seropositivity for antibodies specific for avian-origin influenza A (H7N9) among 1129 individuals of the general population, whereas >6% of 396 poultry workers were positive (on the basis of a hemagglutination inhibition titer of ≥ 80) for this subtype, confirming that infected poultry is the principal source of human infections and that subclinical infections are possible. Fourteen days after symptom onset, elevated levels of antibodies to A(H7N9) were found in 65.8% of patients (25/38) who survived but in only 28.6% of those (2/7) who died, suggesting that the presence of antibodies may improve clinical outcome in infected patients. Keywords. influenza; H7N9; H1N1; antibodies; poultry workers; serological surveillance.

Three cases of severe community-acquired pneumonia caused by a novel influenza A(H7N9) were initially identified in Shanghai and Anhui, China, in March 2013 [1]. Since April 2013, sporadic human infections with this emerging virus were

Received 7 June 2013; accepted 12 July 2013; electronically published 9 August 2013. a S. Y. and Y. C. contributed equally to this work. Correspondence: Lanjuan Li, MD, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Rd, Hangzhou, 310003, China ([email protected]). The Journal of Infectious Diseases 2014;209:265–9 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@ oup.com. DOI: 10.1093/infdis/jit430

BRIEF REPORT

•

JID 2014:209 (15 January)

•

265

Downloaded from http://jid.oxfordjournals.org/ by guest on July 13, 2015

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University; 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou; and 3State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China

subsequently recognized in the Yangtze River Delta in eastern China [2]. According to the information released by the National Population and Family Planning Commission of China and the World Health Organization, as of 21 May 2013, a total of 131 human cases with 39 fatalities were laboratory-confirmed in 10 different provinces and municipalities in the mainland [3a], and 1 additional imported case from Jiangsu province was reported in Taiwan [4, 5]. Human infection with influenza A(H7N9) has not been previously reported in China or other countries. While no apparent outbreak caused by avian influenza viruses was observed in poultry in these areas before or during the emergence of influenza A(H7N9) infection in humans, poultry infected with influenza A(H7N9) in wet markets were identified and recognized as the principal source of human infections [6]. Control measures such as closing live poultry markets in affected areas were imposed to minimize human exposure to potential infected sources. No new cases had been reported at the time of writing (Supplementary Figure 1). Phylogenetic analysis revealed that influenza A(H7N9) is a novel reassortant, possibly generated through multiple reassortment events to incorporate the hemagglutinin (HA) gene from H7 subtype influenza virus circulating in domestic ducks in the same region, the N9 neuraminidase (NA) gene from wild birds, and internal genes from avian influenza A(H9N2) [6, 7]. It is currently unclear whether this influenza A(H7N9) strain was circulating in poultry before this outbreak in humans or whether it was recently generated in avian species. While most of the confirmed human cases were clinically severe, mild and asymptomatic cases were also identified in fever clinics, by retrospective examination of archived samples from patients with mild respiratory tract symptoms, and by contact tracing in our investigation and another report [8]. There has been concern that this virus may have been circulating in the general population, thus far causing mostly asymptomatic infections. The major outbreak occurred in April 2013; it appears that the influenza A(H7N9) human outbreak subsided in May, but it is unclear whether this virus may reemerge. It has been a concern that there may be unrecognized cases, but no evidence is available to determine whether and to what extent unrecognized influenza A(H7N9) infection exists in the affected areas. Serological studies provide important parameters to assess prior infections with influenza viruses and to determine immune protection from infection at a population level [9]. In this study, we determined the seroprevalence of antibodies to the influenza A(H7N9) among laboratory-confirmed patients,

poultry workers, and the general population in Zhejiang province, which is the province with the highest number of influenza A(H7N9) human cases in China. We also analyzed the kinetics of antibody production in patients infected with influenza A(H7N9). METHODS

RESULTS Of the 1129 serum samples collected from individuals (age range, 1–88 years) in the general population, 9 (0.8%) had an 266

•


•

Yang et al

DISCUSSION Serological surveillance for influenza A virus–reactive antibodies by use of an HI assay has proved to be a useful tool for the recognition of past infections with seasonal and zoonotic influenza A viruses [11, 12]. In this study, we showed that 6.3% of poultry workers in areas of the 2013 epidemic of influenza A(H7N9) infection were seropositive for antibodies against the emerging influenza A(H7N9), with HI titers of ≥80, whereas no individuals from the general population of the same areas had HI titers of ≥80. This is not unexpected, because a previous study also showed a similar seroprevalence of avian influenza A(H7) among poultry workers in 1998–2003 [13]. Our data support the conclusion that influenza A(H7N9) or a closely related virus is circulating in live poultry markets and that infected poultry is the principal source for human infections [6]. The presence of antibodies against the influenza A(H7N9) among asymptomatic poultry workers suggests that subclinical human infections do occur. However, another serological study of serum specimens collected between January 2012 and November 2012 from poultry workers in Shanghai, Zhejiang, Jiangsu,


This study was approved by the institutional review board of the First Affiliated Hospital, College of Medicine, Zhejiang University. Serum samples were collected between 28 April and 6 May from 1129 individuals in 3 cities in Zhejiang Province and elsewhere where initial human infections with influenza A (H7N9) were identified (Supplementary Figure 1 and Supplementary Table 1). A standardized epidemiological surveillance form was used to collect epidemiologic and clinical data, including demographic characteristics, history of seasonal influenza or 2009 influenza A(H1N1) vaccination, recent exposure to poultry or other animals, recent visits to wet markets, underlying medical conditions, and clinical symptoms and signs (Supplementary Table 1). Serum samples were also collected from 396 subjects with occupational exposures to poultry from 155 live poultry markets located in 10 districts where human cases of influenza A(H7N9) infection were identified (Supplementary Table 2). Nasal swabs were collected from these poultry workers at the time of serum collection. Single or serial serum samples from a total of 45 influenza A(H7N9)–infected patients, in whom infection was confirmed by virus culture and/or reverse-transcription polymerase chain reaction (RTPCR) analysis of respiratory samples, who were hospitalized in the First Affiliated Hospital of Zhejiang University were also included in this survey. Serum samples were prepared using standard procedures and were stored at −80°C until use. Hemagglutinin inhibition (HI) assays for antibodies against influenza A(H7N9) were conducted using a recombinant virus containing HA and NA from A/Zhejiang/DTID-ZJU01/13(H7N9) (GenBank accession nos. KC885956 and KC885958) [6], with the internal gene backbone of A/PR8/34. Antibodies against seasonal circulating influenza A virus were examined for reference, using A/ Zhejiang/ DTIDZJU02/2009(H1N1) (GenBank accession nos. GU112090GU112097). Because influenza A(H7N9) is still an avian influenza virus, turkey red blood cells were used in the HI assay as previously described [10]. RT-PCR specific for the HA of influenza A(H7N9) was performed on the nasal swab samples as previously described [6]. Categorical variables were compared using the χ2 test.

HI titer of ≥ 40 to influenza A(H7N9), but no serum samples with an HI titer of ≥ 80 were found (Table 1). In contrast, among poultry workers, 13.9% (55/396) and 6.3% (25/396) had influenza A(H7N9) antibody titers of ≥40 and ≥80 (20 had an HI titer of 80, and 5 had an HI titer of 160), respectively. There was a significant difference in the rate of influenza A(H7N9) HI antibody titers of ≥80 between poultry workers and the general population (P < .0001). The viral genome of influenza A (H7N9) was not detected by RT-PCR in any of the nasal swabs collected from these poultry workers. There was no statistically significant difference in the number of subjects with an influenza A(H1N1) HI antibody titer of ≥80 between members of the general population and poultry workers (37.8% and 38.1%, respectively; P = .930). Among the influenza A(H7N9)–seropositive (HI titer, ≥ 80) and influenza A(H7N9)–seronegative poultry workers (HI titer, ≤ 40), there was no sex- or age-specific difference. Among the 45 hospitalized patients with laboratoryconfirmed influenza A(H7N9) infection, 38 survived; 25 (65.8%) who survived had an HI antibody titer of ≥80 for influenza A (H7N9) from as early as 10 days after onset of disease symptoms (Figure 1 and Supplementary Table 1B). Among 20 patients for whom serial serum samples were available, the mean interval between symptom onset and serum conversion (HI titer, ≥80) was 13.5 days (Figure 1). Among 7 fatal cases, only 28.6% (2) had an HI titer of ≥80 in convalescent-phase serum samples collected >14 days after hospitalization (Figure 1 and Table 1). Neutralization assay with both positive and negative serum specimens found that HI titers correlated well with neutralization titers (data not shown).

Table 1. Antibody Titers to the 2013 influenza A(H7N9) and 2009 H1N1 Viruses Infected Patients, Poultry Workers and General Population in the influenza A(H7N9) Affected Areas in China Ab titer to 2009 Influenza A(H1N1), Subjects, No. (%)b

Ab Titer to Influenza A(H7N9), Subjects, No. (%)a Subjects General population (n = 1129) Poultry workers (n = 396) Patients with laboratory-confirmed infection (n = 45)

80

≥40

80 207 (18.3) 79 (20.1) 12 (18.3)

Antibody titers were determined by hemagglutination inhibition (HI) assay, as described previously. Detailed information for all subjects is provided in Supplementary Tables 1 and 2. a

The influenza A(H7N9) and 2009 A(H1N1) strains used in the HI assays were A/Zhejiang/DTID-ZJU01/13(H7N9) and A/Zhejiang/DTID-ZJU02/2009(H1N1), respectively.

b The influenza A(H7N9) strain used in this study is a recombinant virus containing hemagglutinin and neuraminidase from A/Zhejiang/DTID-ZJU01/13 and internal genes from A/PR8/34.

There was a significant difference in the rate of positive influenza A(H7N9) HI antibody titers (defined as a titer of ≥80) between poultry workers and the general population (P < .0001).

c

There was no significant difference in the rate of positive influenza A(H1N1) HI antibody titers (defined as a titer of ≥80) between poultry workers and the general population (P = .930).

d

difference in seroprevalence of antibodies to seasonal 2009 influenza A(H1N1) between the poultry workers and general population involved in this study (Table 1), suggesting that

Figure 1. Spectrum of antibodies to influenza A(H7N9) as estimated by hemagglutinin inhibition (HI) assays along the course of disease. Mean values of HI antibody titers in serum specimens from patients who survived (n = 38) and patients who died (n = 7) were plotted. The dotted line represents average antibody titers for patients who died, and the solid line represents average antibody titers for patients who survived. Logarithmic transformation of HI antibody titers was used for significance testing. There was a significant difference in HI antibody titers between patients who survived and those who died on or after day 21 after symptom onset (P < .05). BRIEF REPORT

•


•

267


and Anhui provinces found no evidence of seropositivity [14], suggesting that the presence of antibodies to influenza A(H7N9) among poultry workers is a recent event. Our data showed no

268

•


•

Yang et al

convalescent-phase plasma specimens, and hyperimmune immunoglobulins containing antibodies to this virus as therapeutic options for severe cases are urgently needed [17]. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes Acknowledgments. We thank the staff of the clinical laboratories and Department of Infectious Disease at the First Affiliated Hospital, Zhejiang University, and the staff of State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, University of Hong Kong, for their contributions. Financial support. This work was supported by the National Emergency Program for Avian Influenza H7N9 of China (KJYJ-2013-01-01), the National Natural Science Foundation of China (81001271), and Mega-projects of Science Research for the 12th Five-Year Plan of China (2013ZX10004901, 2013ZX10004904, and 2012ZX10004-210). H. C. was partly supported by the Area of Excellence of the University Grant Committee (grant AoE/M-12/06). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References 1. Gao R, Cao B, Hu Y, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 2013; 368:1888–97. 2. Li Q, Zhou L, Zhou M, et al. Preliminary report: epidemiology of the avian influenza A (H7N9) outbreak in China. [published online ahead of print April 24 2013]. N Engl J Med 2013. doi:10.1056/NEJMoa 1304617. 3. http://www.who.int/influenza/human_animal_interface/influenza_h7n9/ WHO_H7N9_review_31May13.pdf. 4. http://www.who.int/csr/don/2013_04_25/en/index.html. 5. Chang SY, Lin PH, Tsai JC, Hung CC, Chang SC. The first case of H7N9 influenza in Taiwan. Lancet 2013; 381:1621. 6. Chen Y, Liang W, Yang S, et al. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 2013; 381:1916–25. 7. Liu D, Shi W, Shi Y, et al. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 2013; 381:1926–32. 8. Xu C, Havers F, Wang L, et al. Monitoring avian influenza A(H7N9) virus through national influenza-like illness surveillance, China. Emerg Infect Dis 2013. 9. Zhang AJ, To KK, Tse H, et al. High incidence of severe influenza among individuals over 50 years of age. Clin Vaccine Immunol 2011; 18:1918–24. 10. Wu WL, Chen Y, Wang P, et al. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007. J Virol 2008; 82:1798–807. 11. Wang TT, Parides MK, Palese P. Seroevidence for H5N1 influenza infections in humans: meta-analysis. Science 2012; 335:1463. 12. Boni MF, Chau NV, Dong N, et al. Population-level antibody estimates to novel influenza A/H7N9. J Infect Dis 2013; 208:554–8.


these people were similarly exposed to the circulating seasonal influenza virus strain(s). Although avian-origin influenza A (H7N9) caused 132 laboratory-confirmed human infections within the space of 1 month (Supplementary Figure 1), a lack of antibodies in the general population may indicate that these cross-species transmissions to humans are recent and sporadic events and that the ability of the current influenza A(H7N9) strain for human-to-human transmission is limited at this stage. Active surveillance for infected and suspected human cases in affected areas in China may have played a critical role in blocking further transmissions of influenza A(H7N9) to humans since May 2013 (Supplementary Figure 1). It is also possible that the antibodies to influenza A(H7N9) detected among poultry workers in this study may have resulted from exposure to other antigenically similar avian influenza A(H7) viruses [12], such as influenza A(H7N3), was recently reported in ducks in the region [15]. Further surveillance of avian influenza A(H7) in domestic poultry in China is warranted. Induction of antibodies during severe human infections caused by avian-origin influenza virus, such as H5N1, has not been clearly studied. During this epidemic of avian-origin influenza A(H7N9), Zhejiang Province reported the highest number of hospitalized cases, 45, of whom 7 died. These laboratory-confirmed influenza A(H7N9)–infected patients provided a unique opportunity to assess the kinetics of antibody induction during virus infection and disease recovery. Our data showed that 65.8% of influenza A(H7N9)–infected patients who survived (25/38) developed an HI antibody titer of ≥80 to influenza A(H7N9) soon after symptom onset, while only 28.6% of the patients who died (2/7) generated antibody titers of ≥80. While further studies involving more subjects are needed, it seems that antibody responses are associated more strongly with surviving cases, rather than fatal cases. A previous study of 2009 pandemic influenza A(H1N1)–infected patients showed higher titers of neutralizing antibody with higher avidity in patients with more-severe disease [16]. Further studies are needed to understand the antibody response in influenza A(H7N9)–infected patients. After the initial outbreak in eastern China, humans infected with influenza A(H7N9) were identified in Beijing and some northern and southern provinces of China. It is suspected that the influenza A(H7N9) may have become established in poultry and could reemerge in humans. Preparation and evaluation of human vaccines against this virus should be considered. Our findings on the kinetics of antibody induction among hospitalized influenza A(H7N9)– infected patients seems to also suggest that influenza A(H7N9) is relatively immunogenic and that a vaccine could be effective in preventing human infection and disease in the future. To prepare for the possible reemergence or progression of the epidemic by means of influenza A(H7N9) or a variant of this virus, studies of virus-specific therapeutic antibodies,

13. Puzelli S, Di Trani L, Fabiani C, et al. Serological analysis of serum samples from humans exposed to avian H7 influenza viruses in Italy between 1999 and 2003. J Infect Dis 2005; 192:1318–22. 14. Bai T, Zhou J, Shu Y. Serologic study for influenza A (H7N9) among high-risk groups in China. N Engl J Med 2013; 368:2339–40. 15. Hai-bo W, Ru-feng L, En-kang W, et al. Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in China in 2011. Arch Virol 2012; 157:2017–21.

16. To KK, Zhang AJ, Hung IF, et al. High titer and avidity of nonneutralizing antibodies against influenza vaccine antigen are associated with severe influenza. Clin Vaccine Immunol 2012; 19:1012–8. 17. Hung IF, To KK, Lee CK, et al. Hyperimmune intravenous immunoglobulin treatment: a multicentre double-blind randomized controlled trial for patients with severe A(H1N1)pdm09 infection. Chest 2013; 144:464–73.


BRIEF REPORT

•


•

269