Ahead of Print -Influenza A(H1N1)pdm09 Virus Infection in Giant Pandas, China - Volume 20, Number 3—March 2014 - Emerging Infectious Disease journal - CDC

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Ahead of Print -Influenza A(H1N1)pdm09 Virus Infection in Giant Pandas, China - Volume 20, Number 3—March 2014 - Emerging Infectious Disease journal - CDC

Volume 20, Number 3—March 2014

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Influenza A(H1N1)pdm09 Virus Infection in Giant Pandas, China

Article Contents

• The Study
• Conclusions
• Acknowledgments
• References
• Figure 1
• Figure 2
• Table
• Technical Appendix - 198 KB 
• Suggested Citation

Desheng Li, Ling Zhu, Hengmin Cui, Shanshan Ling, Shengtao Fan, Zhijun Yu, Yuancheng Zhou, Tiecheng Wang, Jun Qian, Xianzhu Xia, Zhiwen Xu, Yuwei Gao1 , and Chengdong Wang1

Author affiliations: Key Laboratory of Animal Disease and Human Health, College of Veterinary Medicine of Sichuan Agricultural University, Ya’an, People’s Republic of China (D. Li, L. Zhu, H. Cui, Z. Xu); Key Laboratory of Animal Biotechnology Center of Sichuan Province, College of Veterinary Medicine of Sichuan Agricultural University, Ya’an (D. Li, L. Zhu, Y. Zhou, Z. Xu, C. Wang); China Conservation and Research Center for the Giant Panda, Ya’an (D. Li, S. Ling, C. Wang); Research Center of Wildlife Disease, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute of Academy of Military Medical Sciences, Changchun, People’s Republic of China (T. Wang, J. Qian, X. Xia, Y. Gao); Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College, Beijing, People’s Republic of China (S. Fan, Z. Yu)

Suggested citation for this article

Abstract

We confirmed infection with influenza A(H1N1)pdm09 in giant pandas in China during 2009 by using virus isolation and serologic analysis methods. This finding extends the host range of influenza viruses and indicates a need for increased surveillance for and control of influenza viruses among giant pandas.

In April 2009, the Centers for Disease Control and Prevention reported the emergence of a novel strain of influenza A(H1N1) virus, which is now referred to as influenza A(H1N1)pdm09 or pH1N1. This virus rapidly affected countries worldwide and continues to circulate as a seasonal influenza virus. In addition to widespread infection of humans, reported have been published of pH1N1 virus infection in domestic and nondomestic animals, including cats, dogs, ferrets, swine, and several wildlife species (1–4). Here, we report a confirmed case of pH1N1 virus infection in giant pandas (Ailuropoda melanoleuca) in China.

The Study

During the human outbreak of pH1N1 in China, 3 giant pandas at the Conservation and Research Center for the Captive Giant Panda in Ya’an City, Sichuan Province, showed clinical signs suggestive of a respiratory tract infection, including pyrexia, anorexia, malaise, conjunctivitis, and sneezing. Nasal swab specimens were successfully collected under anesthesia from 1 of the affected pandas (Ximeng). All 3 pandas recovered after receiving 75 mg of oseltamivir phosphate twice daily for 5–6 days. For serologic analysis, serum samples were collected from all 3 pandas ≈3 months after the resolution of the respiratory illness; a serum sample collected before onset of the respiratory illness was also available for all 3 animals.

The nasal swab specimens were collected in 1 mL phosphate-buffered saline and tested for evidence of pH1N1 virus and several other pathogens reported (5–7) or suspected to occur in giant pandas: canine distemper virus, canine adenovirus, canine coronavirus, canine herpesvirus, and canine parainfluenza virus. Testing for detection of influenza A virus was performed by using a real-time reverse transcription PCR method, as described by the World Health Organization (WHO) (8); other pathogens were tested by different PCR methods.

RNA from the swab specimens tested positive for the hemagglutinin gene of pH1N1 virus. No other pathogens were detected.

Figure 1

Figure 1. . Negative-staining electron micrograph image showing influenza A(H1N1)pdm09 virus particles (arrowheads) in allantoic fluid supernatant collected from specific pathogen free eggs after injection with a nasal swab sample collected from a...

To isolate and characterize the pH1N1 virus, we injected 10-day-old specific pathogen free embryonated chicken eggs with material collected from one of the nasal swab samples. Allantoic fluid from the injected eggs agglutinated 0.5% (vol/vol) chicken erythrocytes, indicating the presence of replication-competent virus in the nasal swab sample. When evaluated by electron microscopy, allantoic fluid supernatant from the infected egg displayed enveloped influenza virus–like particles of 100–120 nm (Figure 1).

Figure 2

Figure 2. . Phylogenetic trees of influenza A(H1N1)pdm09 virus (pH1N1) isolated from a giant panda in China compared with previously characterized pandemic influenza A(H1N1) viruses. A) Hemagglutinin gene nucleotide sequences; B) neuraminidase gene...

We sequenced the entire genome of the virus by using RNA harvested from the allantoic fluid of infected eggs. Sequence analysis was performed as described by WHO by using methods for pH1N1 virus (9). A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify sequences similar to those of the giant panda isolate; these sequences were GenBank accession nos. KF277197–KF277204. Analyses showed that each of the 8 gene segments of the virus we isolated were closely related to pH1N1 viruses circulating among humans, including a human representative strain (A/California/04/2009) and a contemporary strain (A/Sichuan/1/2009); these viruses showed 98.6%–100% nt identity to the panda strain (Technical Appendix  [PDF - 198 KB - 2 pages]Table 1). Phylogenetic analysis of the 8 gene segments of the virus we isolated showed that the isolate was related to the pH1N1 virus clade of influenza viruses (Figure 2; Technical Appendix  [PDF - 198 KB - 2 pages] Figure). The results of the BLAST and phylogenetic analyses suggest that a human pH1N1 virus was transmitted directly to giant pandas without recombination or significant adaptation. We named the virus that we isolated A/giant panda/01/Ya’an/2009 (H1N1).