Influenza A(H1N1)pdm09 Virus Infection in Giant Pandas, China
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.
During the human outbreak of pH1N1 in China, 3 giant pandas at the Conservation and Research Center for the 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.
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).
Serum samples collected from the affected pandas before their illnesses and 3 months after the resolution of the illnesses were tested for the presence of hemagglutination inhibition (HI) antibodies against a panel of influenza A viruses. HI antibody titers were measured against the A/giant panda/01/Ya’an/2009 (H1N1) isolate, human seasonal influenza (H3N2) virus (A/Victoria/361/2011), avian (H5N1) virus (A/duck/Anhui/1/2006), avian (H6N1) virus (A/Mallard/SanJiang/275/2007), and avian (H7N1) virus (A/Baer’s pochard/HuNan/414/2010). All 3 pandas had high titers of HI antibodies against the pH1N1 virus 3 months after illness (Table).
One panda (Zhangka) had detectable pH1N1 and H3 subtype HI antibodies before infection; this serum sample was collected before the display of overt clinical signs in the animal but after the onset of the human pH1N1 outbreak in China. It is unknown if these antibodies reflect previous exposure to pH1N1 virus or are reflective of cross-reactive antibodies generated against antigenic sites in previously circulating influenza viruses.
Two of the pandas (Gege and Zhangka) also had serum antibodies that inhibited hemagglutination mediated by an H6 subtype avian influenza virus 3 months after the respiratory infection, whereas no such antibodies were detected before the respiratory infection (Table). When reference serum samples known to contain HI antibodies against each of the viral subtypes were evaluated for potential cross-reactivity against the other influenza subtypes, we observed no apparent cross-reactivity of pH1N1 antibodies against the H6 subtype virus (Technical Appendix [PDF - 198 KB - 2 pages] Table 2). Therefore, although it is possible that the high titers of HI antibodies against pH1N1 virus in these animals cross-reacted with the H6 subtype viruses, we cannot exclude the possibility that these antibodies were generated in response to an independent exposure to an H6 influenza virus.
Influenza A(H1N1)pdm09 virus infection has been found in mammals and birds of a variety of species since the virus was identified in early 2009 (1–4). We documented infection with this virus in giant pandas in China during 2009. Sequence analysis of a virus isolated from a nasal swab sample revealed that it was nearly identical to the pH1N1 virus circulating in humans, which suggests that the virus may have been transmitted to the pandas directly from humans. Furthermore, serologic analysis of samples from the affected pandas was consistent with productive infection by pH1N1 virus. Seroconversion was documented in each animal, although 1 animal appeared to have been previously exposed to pH1N1 or a related virus that was able to elicit cross-reactive antibodies. Similarly, some pandas had detectable HI antibodies against H3 and H6 subtype influenza viruses, raising the possibility that giant pandas may be infected with human- and avian-origin influenza viruses.
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- 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...
- 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)...
- Table. Hemagglutination inhibition antibody titers against influenza A viruses in serum samples collected from giant pandas before and after respiratory infection, China, 2009
- Technical Appendix. . Nucleotide identity of influenza A(H1N1)pdm09 virus isolated from a giant panda compared with other influenza viruses; antibody titers of reference serum sample against influenza viruses of different subtypes;... 198 KB