J Vet Sci.  2009 Sep;10(3):261-263. 10.4142/jvs.2009.10.3.261.

Isolation and identification of a canine coronavirus strain from giant pandas (Ailuropoda melanoleuca)

• Gao FS
• Hu GX
• Xia XZ
• Gao YW
• Bai YD
• Zou XH

• KMID: 1726927
• DOI: http://doi.org/10.4142/jvs.2009.10.3.261

Abstract

Two giant pandas (Ailuropoda melanoleuca) died of unknown causes in a Chinese zoo. The clinical disease profile suggested that the pandas may have suffered a viral infection. Therefore, a series of detection including virus isolation, electron microscopy, cytobiological assay, serum neutralization and RT-PCR were used to identify the virus. It was determined that the isolated virus was a canine coronavirus (CCV), on the basis of coronavirus, neutralization by canine anti-CCV serum, and 84.3% to 100% amino acid sequence similarity with CCV. The results suggest that the affected pandas had been infected with CCV.

Keyword

canine coronavirus; giant panda; virus characterization; virus isolation

MeSH Terms

Amino Acid Sequence
Animal Diseases/*virology
Animals
Animals, Zoo/*virology
Coronaviridae Infections/*veterinary/virology
Coronavirus, Canine/genetics/*isolation & purification
Fatal Outcome
Female
Male
Molecular Sequence Data
Sequence Alignment
Sequence Homology, Amino Acid
Ursidae/*virology
Viral Proteins/chemistry

Figure

• 

  Fig. 1 Isolation and culture of giant panda virus (GPV) in Felis catus whole fetus (FCWF) cell line. (A) Uninfected FCWF cells; (B) FCWF cells inoculated with 10,000 TCID50 GPV. The arrows indicate cells with cytopathic effect, which became round and detatch from the bottom of the culture flask. ×400.

• 

  Fig. 2 Detection of GPV under electron microscopy. (A) Detection of GPV particles in culture supernatant by negative staining. The arrows indicate the corona spikes of the GPV particles. (B) Detection of inclusion bodies of GPV in FCWF cells by ultrathin section. The white arrow indicates the cytoblast of an infected FCWF cell. The black arrow indicates the inclusion bodies of GPV in the cytoplasm of cells. Scale bars = 200 nm.

• 

  Fig. 3 Analysis of GPV based on gene sequencing. (A) Comparison of amino acid sequences of S gene product from GPV by nested PCR assay with that of the S gene of canine coronavirus (CCV) K378 (X77047). The asterisk indicates conserved amino acids between GPV and CCV K378; the dot indicates synonymous mutations of amino acids between GPV and CCV K378; the blanks indicate mutant amino acids between GPV to CCV K378; the number after the virus' name corresponds to the amino acid position in the S gene. (B) Percentage of amino acid identity between the partial S gene of GPV and CCV.

Reference

1. Glass RT, Bullard JW, Conrad RS, Blewett EL. Evaluation of the sanitization effectiveness of a denture-cleaning product on dentures contaminated with known microbial flora. An in vitro study. Quintessence Int. 2004. 35:194–199.

2. Keenan KP, Jervis HR, Marchwicki RH, Binn LN. Intestinal infection of neonatal dogs with canine coronavirus 1-71: studies by virologic, histologic, histochemical, and immunofluorescent techniques. Am J Vet Res. 1976. 37:247–256.

3. Loeffler IK, Howard J, Montali RJ, Hayek LA, Dubovi E, Zhang Z, Yan Q, Guo W, Wildt DE. Serosurvey of ex situ giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China with implications for species conservation. J Zoo Wildl Med. 2007. 38:559–566.

4. Mainka SA, Qiu X, He T, Appel MJ. Serologic survey of giant pandas (Ailuropoda melanoleuca), and domestic dogs and cats in the Wolong Reserve, China. J Wildl Dis. 1994. 30:86–89.

5. Naylor MJ, Harrison GA, Monckton RP, McOrist S, Lehrbach PR, Deane EM. Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate. J Clin Microbiol. 2001. 39:1036–1041.

6. Nermut MV. Negative staining of viruses. J Microsc. 1972. 96:351–362.

7. Roche RR, Alvarez M, Guzman MG, Morier L, Kouri G. Comparison of rapid centrifugation assay with conventional tissue culture method for isolation of dengue 2 virus in C6/36-HT cells. J Clin Microbiol. 2000. 38:3508–3510.

8. Tang G, Tang X, Gan D. A method of preparing the section of eye tissue for transmission electron microscopy. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 1999. 16:237–239.

9. Woods RD, Wesley RD. Immune response in sows given transmissible gastroenteritis virus or canine coronavirus. Am J Vet Res. 1986. 47:1239–1242.

10. Zhang JS, Daszak P, Huang HL, Yang GY, Kilpatrick AM, Zhang S. Parasite threat to panda conservation. Ecohealth. 2008. 5:6–9.