Study of cultural properties of canine enteric coronavirus isolate in different cell cultures

The etiology of the diseases affecting gastrointestinal tractofdogs is variable. The second most important enteric viral pathogen in the world after parvovirus is coronavirus (CCoV). Vast studies by scientists from different countries evidence the ubiquitous spread of coronavirus infection in dog populations. In this regard, the prevention of this disease is still an acute problem; firstly this means the development of effective vaccines, which can provide are liable protection from the infection. The aim of this work was to study the CCoV isolate cultural properties, the selection of cell cultures most suitable for its reproduction and optimization of virus cultivation parameters in sensitive cell lines. TheCCoV isolate recovered from the pathological material of a dead puppy with enteritis symptoms was used in the study. Seven continuousandtwoprimarytrypsinizedcellculturesweretestedduringthestudyanditwasestablishedthat the most sensitive cell culture for the reproduction of this isolate was continuous Crandell-Rees Feline Kidney Cells (CRFK) in which atypical cytopathic effect was notedon Day 2 of the cultivation. Virus infectivity titer in this culture was 3.58 ± 0.14 lgTCID50/cm3 . It was established that such cell linesas fel in espleen cells (FS) and primary trypsinized kitten spleen cells (KS) are also sensitive to the CCoV isolate but less than CRFK cells. Effect of such parameters as multiplicity of infection, cultivation time, inoculation technique, adsorption period and cell lineag eon coronavirus growth rate was studiedfor some selected cell cultures. It was concluded that CRFK and FS cells can be used for the propagation of viral material to develop diagnostic tools and vaccines against canineenteric coronavirus.

1. Alves C. D. B. T., Granados O. F. O., Budaszewski R. D. F., Streck A. F., Weber M. N., Cibulski S. P., et al. Identification of enteric viruses circulating in a dog population with low vaccine coverage. Braz. J. Microbiol. 2018; 49 (4): 790–794. DOI: 10.1016/j.bjm.2018.02.006.

2. McElligott S., Collins P. J., Sleator R. D., Martella V., Decaro N., Buonavoglia C., O’Shea H. Detection and genetic characterization of canine parvoviruses and coronaviruses in southern Ireland. Arch. Virol. 2011; 156 (3): 495–503. DOI: 10.1007/s00705-010-0861-3.

3. Decaro N., Desario C., Billi M., Mari V., Elia G., Cavalli A., et al. Western European epidemiologicalsurvey for parvovirus and coronavirusinfections in dogs. Vet. J. 2011; 187 (2): 195–199. DOI: 10.1016/j.tvjl.2009.10.027.

4. Decaro N., Buonavoglia C. An update on canine coronaviruses: viral evolution and pathobiology. Vet. Microbiol. 2008; 132 (3–4): 221–234. DOI: 10.1016/j.vetmic.2008.06.007.

5. MaG. G., Lu C. P. Two genotypes of canine coronavirussimultaneously detected in the fecalsamples of healthy foxes and raccoon dogs. Wei Sheng Wu Xue Bao. 2005; 45 (2): 305–308. PMID: 15989282. (in Chinese)

6. Rosa G. M., Santos N., Grøndahl-Rosado R., Fonseca F. P., Tavares L., Neto I., et al. Unveiling patterns of viral pathogen infection in free-ranging carnivores of northern Portugal using a complementary methodological approach. Comp. Immunol. Microbiol. Infect. Dis. 2020; 69:101432. DOI: 10.1016/j.cimid.2020.101432.

7. Zarnke R. L., Evermann J., Ver Hoef J. M., McNay M. E., Boertje R. D., Gardner C. L., et al. Serologic survey for canine coronavirus in wolves from Alaska. J. Wildl. Dis. 2001; 37 (4): 740–745. DOI: 10.7589/0090-3558-37.4.740.

8. Sanchez-Morgado J. M., Poynter S., Morris T. H. Molecular characterization of a virulent canine coronavirus BGF strain. Virus Res. 2004; 104 (1): 27–31. DOI: 10.1016/j.virusres.2004.02.038.

9. Evermann J. F., Abbott J. R., Han S. Canine coronavirus-associated puppy mortality without evidence of concurrent canine parvovirus infection. J. Vet. Diagn. Invest. 2005; 17 (6): 610–614. DOI: 10.1177/1040638705 01700618.

10. Escutenaire S., Isaksson M., Renström L. H., Klingeborn B., Buonavoglia C., Berg M., et al. Characterization of divergent and atypical canine coronaviruses from Sweden. Arch. Virol. 2007; 152 (8): 1507–1514. DOI: 10.1007/s00705-007-0986-1.

11. Buonavoglia C., Decaro N., Martella V., Elia G., Campolo M., Desario C., et al. Canine coronavirus highly pathogenic for dogs. Emerg. Infect. Dis. 2006; 12 (3): 492–494. DOI: 10.3201/eid1203.050839.

12. Zicola A., Jolly S., Mathijs E., Ziant D., Decaro N., Mari V., Thiry E. Fatal outbreaks in dogs associated with pantropic canine coronavirus in France and Belgium. J. SmallAnim. Pract. 2012; 53 (5): 297–300. DOI: 10.1111/j.1748- 5827.2011.01178.x.

13. Ntafis V., Mari V., Danika S., Fragkiadaki E., Buonavoglia C. An outbreak of canine coronavirusin puppiesin aGreek kennel. J. Vet. Diagn. Invest. 2010; 22 (2): 320–323. DOI: 10.1177/104063871002200231.

14. Radford A. D., Singleton D. A., Jewell C., Appleton C., Rowlingson B., Hale A. C., et al. Outbreak of severe vomiting in dogs associated with a canine enteric coronavirus, United Kingdom. Emerg. Infect. Dis. 2021; 27 (2): 517–528. DOI: 10.3201/eid2702.202452.

15. Collins M., Singleton D. A., Noble P. J. M., Pinchbeck G. L., Smith S., Brant B., et al. Small animal disease surveillance 2020/21: SARS-CoV-2, syndromic surveillance and an outbreak of acute vomiting in UK dogs. Vet. Rec. 2021; 188 (8):304. DOI: 10.1002/vetr.427.

16. Pratelli A., Elia G., Martella V., Tinelli A., Decaro N., Marsilio F., et al. M gene evolution of canine coronavirusin naturally infected dogs. Vet. Rec. 2002; 151 (25): 758–761. PMID: 12521247.

17. Wang X., Li C., Guo D., Wang X., Wei S., Geng Y., et al. Co-circulation of canine coronavirusI and IIa/b with high prevalence and genetic diversity inHeilongjiang Province, Northeast China. PLoSOne. 2016; 11 (1):e0146975. DOI: 10.1371/journal.pone.0146975.