Pandemic (H1N1) 2009 in Captive Cheetah

Beate Crossley

, Sharon Hietala, Tania Hunt, Glenn Benjamin, Marie Martinez, Daniel Darnell, Adam Rubrum, and Richard Webby

Author affiliations: California Animal Health and Food Safety Laboratory, University of California, Davis, California, USA (B. Crossley, S. Hietala); Safari West, Santa Rosa, California, USA (T. Hunt, G. Benjamin, M. Martinez); St. Jude Children’s Research Hospital, Memphis, Tennessee, USA (D. Darnell, A. Rubrum, R. Webby)

Abstract

We describe virus isolation, full genome sequence analysis, and clinical pathology in ferrets experimentally inoculated with pandemic (H1N1) 2009 virus recovered from a clinically ill captive cheetah that had minimal human contact. Evidence of reverse zoonotic transmission by fomites underscores the substantial animal and human health implications of this virus.

Case Report

In November 2009, during the surge of pandemic (H1N1) 2009 cases among humans and ≈7 months after A/California/04/2009(H1N1) was first reported in a child in southern California (1), nasal swab specimens from a cheetah (Acinonyx jubatus) were submitted to the California Animal Health and Food Safety Laboratory. The 8-year-old cheetah, referred to as animal D, belonged to a privately operated wild animal park in northern California. The animal park veterinarian collected the specimens from animal D for diagnostic testing after severe respiratory symptoms, ptyalism, anorexia, and lethargy were observed in the animal and 3 other cheetahs housed in the same area. The 4 animals had been showing clinical signs of an influenza-like illness the previous week and were being treated with a combination of amoxicillin, enrofloxacin, famotidine, and omeprazole.
At the recommendation of a volunteer worker who was professionally affiliated with the California Public Health Department, a nasal swab sample was obtained from animal D 2–3 days following the onset of clinical signs. The California Public Health Department supplied a sampling kit consisting of cotton-tipped swabs, viral transport media, and a shipping container. A swab sample also was obtained from animal C, another 8-year-old cheetah, ≈4–6 days after it showed clinical signs. At the time of sampling, the remaining 2 cheetahs (animals A and B), which were housed in a separate but conjoining area, had clinically recovered from their respiratory illness. To avoid the additional handling and sedation required for sample collection from the nondomesticated animals, park personnel decided not to collect samples from the recovered cats.
The nasal swab samples were processed according to a standardized procedure distributed by the United States Department of Agriculture National Veterinary Services Laboratory through the National Animal Health Laboratory Network. The protocol used was an approved deviation of the standard operating procedure used for testing swine in the United States. In brief, RNA was recovered by using the MagMAX Viral RNA Isolation Kit (Applied Biosystems, Austin, TX, USA) following the manufacturer’s recommendations. Real-time reverse transcription PCR (qRT-PCR) individually targeting the influenza A matrix (M) gene and the neuraminidase (N1) gene were performed as described (2). Positive qRT-PCR test results, with cycle thresholds of 26.83 and 30.23 for the M and N1 genes, respectively, were obtained for animal D. PCR results were confirmed as pandemic (H1N1) 2009 virus by sequence analysis of the PCR amplicon. Animal C had negative test results for M and N1 genes.
A second aliquot of the nasal sample from animal D was inoculated onto trypsin-treated MDCK cells (3), and the propagated cytolytic virus was forwarded to St. Jude Children’s Research Hospital for complete genome sequence analysis. The influenza virus isolate was inoculated into ferrets for further characterization of the virus. The United States Department of Agriculture National Veterinary Services Laboratory (Ames, Iowa, USA) was additionally provided an aliquot of the nasal swab specimen. Sequence analyses of the M, N1, and hemagglutinin genes were performed (GenBank accession nos. HMO12479, HMO 12480, and HMO12481) and confirmed the initial diagnostic detection of pandemic (H1N1) 2009 virus.
Whole genome sequencing was performed by using methods recommended by the World Health Organization (www.who.int/csr/resources/publications/swineflu/sequencing_primers/en/

). Sequence analysis confirmed the existence of the pandemic (H1N1) 2009 virus (A/Cheetah/California/D0912239/2009, GenBank accession nos. CY092750–CY092757). No unique molecular signatures were detected in the cheetah virus as compared with other pandemic subtype H1N1 viruses isolated from humans. Except for 1 synonymous base-pair mismatch at position 1440 in the hemagglutinin gene, the sequence data matched in the 3 genes sequenced in 2 locations.
To characterize and further assess the pathogenicity profile of the virus, we inoculated 3 groups of 5 ferrets each intranasally with a 10^5.5 50% tissue culture infectious dose of A/Cheetah/California/D0912239/2009 or one of the representative strains from humans, A/Tennessee/1–560/09 and A/Ukraine/N6/2009. Animal studies were approved by the St. Jude Animal Care and Use Committee (protocol 428) and were conducted according to applicable laws and guidelines. Ferrets were observed daily, and temperature and weight measurements and nasal washes were obtained for analysis on day 0 and on postexposure days 3, 5, and 7. Weight measurements were performed until day 14.