Norovirus in Captive Lion Cub (Panthera leo)

African lions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including feline calicivirus infection. We report the identification of a novel enteric calicivirus, genetically related to human noroviruses of genogroup IV, in a lion cub that died of severe hemorrhagic enteritis.

African lions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including feline calicivirus infection. We report the identifi cation of a novel enteric calicivirus, genetically related to human noroviruses of genogroup IV, in a lion cub that died of severe hemorrhagic enteritis.
L ions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including infections with canine distemper virus, feline parvovirus, feline retroviruses, feline herpesvirus, and feline calicivirus (FCV) (1)(2)(3)(4). Antibodies to FCV have been detected in captive lions (2), and calicivirus-like particles have been detected in oral vesicular lesions of captive immature lions (3). Despite the presence of FCV-specifi c antibodies and the observation that cub survival may be reduced during calicivirus outbreaks, clear signs of FCV-induced illness have not been described in free-ranging lions (4). We detected a novel calicivirus in a 4-week-old lion cub that died of severe hemorrhagic enteritis.

The Study
In autumn 2004, the Zoo of Pistoia, Italy, adopted 2 adult lions that had been born in captivity. In October 2005 and May 2006, the female gave birth to 2 cubs each delivery, which 3-4 weeks later showed signs of enteritis and died. In October 2006, she gave birth to a single cub, which died of severe hemorrhagic enteritis at 4 weeks of age. The cub exhibited anorexia, depression, and mild dehydration, but it was not moved away from the mother for ethologic and management reasons. In the subsequent days, the cub's general condition appeared to worsen; anorexia and more marked depression were reported by the animal caretakers. Therefore, on day 3 after illness onset, the cub was taken to the zoo's animal hospital. Examination showed a temperature of 38.6°C, hemorrhagic enteritis, tenesmus, and deep sensorial depression. Hydration and antimicrobial therapy were immediately started, but after 24 hours the animal was agonal and hypothermic and was therefore euthanized. At necropsy, severe hemorrhagic enteritis, hemorrhage in the intestinal lymph nodes, and marked dehydration were observed. Histologic examination showed marked alteration of the intestinal mucosa: erosions, villi depletion, and hemorrhagic infi ltration.
The tissues and intestinal contents were screened for common feline and canine viral pathogens by using either conventional or quantitative PCR and reverse transcription-PCR (RT-PCR). Results were negative for known feline (parvovirus, coronaviruses, herpesvirus, retroviruses) and canine (distemper virus, parvovirus, adenoviruses type-1 and type-2) pathogens. Calicivirus was identifi ed in the intestinal content by using a broadly reactive primer pair, p289-p290, targeted to highly conserved motives of the RdRp region of the polymerase complex (5), but unexpectedly, the strain could not be characterized as FCV by using multiple sets of primers specifi c for the FCV capsid gene. In addition, the sample was positive for the norovirus (NoV)-specifi c primer pair JV12Y-JV13I (6).
Bacteriologic investigations detected an Esherichia coli O86, enteropathogenic E. coli (EPEC) group. Clostridium sordelli and C. perfringens were also isolated. By screening of the cpb, cpb2, etx, and cpe genes, the C. perfringens isolate was characterized as toxin-type A.
Sequence analysis of the 315-bp fragment of the RdRp region (strain 387/06) by using BLAST (www.ncbi. nlm.nih.gov/blast) and FASTA (www.ebi.ac.uk/fasta33) showed that the virus was distantly related to FCV (<35% amino acid [aa] identity) but closely related to human and animal NoVs (≤ 75% aa identity). To determine the sequence and genome organization of the novel calicivirus, a 3.4-kb region at the 3′ end of the genome was amplifi ed by RT-PCR as described by Wang et al. (7). The sequence of the 3′ end of open reading frame (ORF)1, the full-length ORF2, ORF3, and the noncoding region through the poly-A tail was determined (GenBank accession no. EF450827). A 14-nt overlap was present in the ORF1-ORF2 junction region, as it is in most human and animal NoVs. The ORF2 was 1,737 nt long and contained an ORF encoding a capsid protein with a predicted size of 578 aa. By BLAST and FASTA analysis, the highest sequence match was found to genogroup IV NoVs (69.3-70.1% aa identity), and identity to non-GIV NoVs was ≤52.6% aa. A total of 23 aa insertions, scattered throughout the P2 domain, were present in the capsid protein of the lion NoV when compared with human genogroup IV NoVs. A 1-nt overlap was found between ORF2 and ORF3, and a 106-nt long nontranslated *University of Bari, Valenzano, Bari, Italy; †Giardino Zoologico di Pistoia, Pistoia, Italy; ‡University of Palermo, Palermo, Italy; §Baranya County Institute of State Public Health Service, Pécs, Hungary; and ¶National Institute of Public Health and the Environment, Bilthoven, the Netherlands region was found between ORF3 and the poly-A tail. ORF3 was 765 nt long and encoded for a 254-aa polypeptide. The nucleotide identity plot of the genome of the lion NoV (from the 3′ end of ORF1 to the poly-A tail) was compared with the human genogroup IV.1 NoV, Fort Lauderdale/560/98/ US (AF414426) (Figure 1). A phylogenic tree was constructed by using the capsid protein of a selection of human and animal NoVs of the various NoV genogroups (I to V) (7,8). In the tree (Figure 2), the lion calicivirus strain was grouped with genogroup IV human NoVs.

Conclusions
NoVs in humans were fi rst discovered by use of electron microscopy in 1972 (10). As a consequence of the development and large-scale application of new and sensitive molecular diagnostic techniques, NoVs are now regarded as the major cause of epidemic, nonbacterial gastroenteritis worldwide in humans of all age groups (9). Human NoVs are classifi ed into genogroups I, II, and IV. In addition, NoVs classifi ed in genogroups II and III have been detected in pigs and cows (7,11,12), and NoVs proposed as genogroup V have been detected in mice (13) (Table). However, to our knowledge, NoVs have not been detected in other animal species and our report is the fi rst description of NoVs in felids.
Because of the possibility of genetic recombination, a consistent and reliable classifi cation of NoV is necessarily based on analysis of the complete capsid gene, and a comprehensive classifi cation scheme has been established by analysis of 164 NoV strains (8). Strains within the same genotype (or cluster) share >85% aa identity; strains of different genotypes within the same genogroup share 55%-85% aa identity (8). The lion NoV 387/06 appeared to be more related genetically to human genogroup IV NoVs (69.3%-70.1% aa identity in the capsid protein). Accordingly, the virus may be considered as a distinct genotype (IV.2) within genogroup IV; human genogroup IV NoVs are genotype IV.1.
The close genetic relationship observed between the lion NoV strain and human genogroup IV NoVs reinforces the notion that the evolution of human NoVs is intermingled with that of animal NoVs. The mechanisms driving the evolution of NoVs are accumulation of punctuated mutations and recombination (14). In addition, NoVs can infect heterologous species, resulting in mild or unapparent infections (15). To assess whether animal NoVs have emerged over time in humans by direct interspecies transmission or by exchange of genetic material through recombination with human NoVs, the genetic diversity of animal NoVs must be explored.
Whether the novel lion calicivirus is a newly identifi ed felid viral pathogen or a NoV strain of heterologous origin detected incidentally in the intestinal content of the cub remains to be proven. Bacterial coinfections were also detected and likely enhanced the severity of the enteritis disease by triggering synergistic effects. Accordingly, the pathogenic potential and the origin of the novel calicivirus strain remain to be elucidated.