Coronavirus infection of spotted hyenas in the Serengeti ecosystem

Abstract

Sera from 38 free-ranging spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, were screened for exposure to coronavirus of antigenic group 1. An immunofluorescence assay indicated high levels of exposure to coronavirus among Serengeti hyenas: 95% when considering sera with titer levels of > or = 1:10 and 74% when considering sera with titer levels of > or = 1:40. Cubs had generally lower mean titer levels than adults. Exposure among Serengeti hyenas to coronavirus was also confirmed by a serum neutralisation assay and an ELISA. Application of RT-PCR to 27 fecal samples revealed viral RNA in three samples (11%). All three positive fecal samples were from the 15 juvenile animals (<24 months of age) sampled, and none from the 12 adults sampled. No viral RNA was detected in tissue samples (lymph node, intestine, lung) from 11 individuals. Sequencing of two amplified products from the S protein gene of a positive sample revealed the presence of coronavirus specific RNA with a sequence homology to canine coronavirus of 76 and 78% and to feline coronavirus type II of 80 and 84%, respectively. Estimation of the phylogenetic relationship among coronavirus isolates indicated considerable divergence of the hyena variant from those in European, American and Japanese domestic cats and dogs. From long-term observations of several hundred known individuals, the only clinical sign in hyenas consistent with those described for coronavirus infections in dogs and cats was diarrhea. There was no evidence that coronavirus infection in hyenas caused clinical signs similar to feline infectious peritonitis in domestic cats or was a direct cause of mortality in hyenas. To our knowledge, this is the first report of coronavirus infection in Hyaenidae.

Fig. 1

Comparison of coronavirus serum antibody titers determined by an indirect immunofluorescence assay for 28 adult hyenas (≥24 months of age) and 7 cubs (≤12 months of age) (Mann–Whitney U-test, U = 139.5, P = 0.08). The central line marks the median titer, each box shows the range of 50% of the values around the median, and the vertical lines indicate the total range of values.

Fig. 2

Phylogenetic relationship between coronavirus isolates from a spotted hyena (HYENA) in the Serengeti National Park, feline enteric coronavirus (X80799), feline infectious peritonitis virus (X06170), canine coronavirus (AF116247, AB017789, AF116245), and the canine coronavirus vaccine strain CCVInSAVC-1 (A22884). The unrooted maximum-likelihood tree (−ln L = 1581.61) based on the 429 bp segment of the 5′ region of the S gene (Posch et al., 2001) was recovered under the best-fit model (HKY + G) selected by hLRTs in Modeltest Version 3.06 (Posada and Crandall, 1998). Base frequencies: A = 0.3042, C = 0.1875, G = 0.1908, T = 0.3175; transition/transversion ratio: 1.6559; gamma distribution shape parameter: 0.2864. Numbers at nodes represent the percentage of 100 bootstrap replicates.