Detection of Coronavirus (CCoV) in Dogs by Transmission Electron Microscopy Techniques

Coronaviruses are known for their ability to cause gastrointestinal, respiratory, and central nervous system diseases in various species of avian, mammalian, and human hosts (Sun et al., 2020). Through an intermediate species, coronaviruses acquire zoonotic potential that allows them to move from a reservoir species to other species, including humans (Cui et al., 2019). This zoonotic ability is evidenced by their genetic plasticity that promotes a high frequency of genetic changes (mutation and recombination) (Decaro & Lorusso, 2020), which also influence tissue tropism and pathogenicity (Vijaykrishna et al., 2007). In dogs, CCoV is one of the most important viral agents causing gastroenteritis. Canine coronavirus is an emerging infectious disease affecting animals of all ages (Kong et al., 2007). Occurring worldwide, it has been detected in various countries, such as the United Kingdom (Radford et al., 2021), Japan (Takano et al., 2015), China (Tian et al., 2021), Turkey (Timurkan et al., 2021), Italy (Zobba et al., 2021), USA (Licitra et al., 2014), and Spain (Decaro et al., 2006). In Brazil, the first outbreak was detected in 1989 by Mitika et al., with viral particles identification by transmission electron microscopy at the Biological Institute of São Paulo, SP, Brazil. Subsequently, other cases were reported (Dezengrini et al., 2007; Castro et al., 2010; Guirao et al., 2013). Canine Enteric Coronavirus (CCoV) belongs to the Alphacoronavirus genus, Coronaviridae family, and Nidovirales order. They are pleomorphic viruses, spherical to elongated, with a characteristic envelope containing surface projections from the viral membrane in the form of a club, solar crown, or petal, measuring 75-160 nm in diameter (Belouzard et al., 2012; Li, 2016). The CCoV genome is single-stranded RNA and measures from 27.6 to 31 kilobases in length. It has four fundamental proteins in its envelope essential for infection. The S glycoprotein is responsible for the entry of infectious virion particles into the target cell through interaction with the host cell receptors, as well as providing the virion with a crown-like appearance. By binding to the nucleocapsid, theM protein acts in viral assembly organization, and theE protein operates in pathogenesis, assembly, and virus release. TheN protein facilitates the interaction of theM protein during virion assembly, promoting increased viral transcription efficiency (Dhama et al., 2020). CCoV has 3 subtypes, types I and IIincluded in the Alphacoronavirus genus, and type III (CRCoV-canine respiratory coronavirus), in the Betacoronavirus genus. Type IIcan be divided into subtypes IIa and recombinant IIb. CCoV types I and IIcause mild asymptomatic enteritis or self-limiting, but pathogenic variants of pantropic II-a CCoV can cause severe disease (Decaro et al., 2012), characterized by loss of appetite, vomiting, profuse bloody and watery diarrhea with a putrid odor, abundant serosanguineous fluid in the abdominal cavity, accompanied by fever, anorexia, vomiting, prostration, and severe dehydration (Hagiwara et al., 1989; Catroxo et al., 1998, Buonavoglia et al., 2006; Zapulli et al., 2008). Mortality is low but can occur, especially in co-infected puppies with parvovirus, canine distemper virus, Ehrlichia canis, Isospora, or other intestinal pathogens (Pratelli et al., 2008; Catroxo et al., 2023). Transmission occurs via the oral-fecal route, through ingestion of contaminated food or water and by direct contact with an infected animal. The incubation period is 1 to 4 days, and the duration of the disease is 2 to 10 days inmost dogs, with cases where symptoms intermittency is observed, with periods of improvement and apparent cure, followed by diarrheal episodes. The viruses reach the duodenum within 48 hours, reach the small intestine, penetrate enterocytes, and begin their replication in the cytoplasm of villous epithelial cells, causing their atrophy. Dogs can be carriers and shed the virus in feces for up to 6 months post-infection (Mitika et al., 1989; Licitra et al., 2014).

Canine coronavirus (CCoV) has a significant impact on veterinary activities in kennels and animal shelters due to the rapid spread of the virus, mainly contaminating young animals aged 2 to 5 months (Decaro & Buonovoglia, 2008), causing economic losses due to mortality and/or morbidity, especially in commercial production kennels (Guirao et al., 2013). This study aimed to detect the presence of coronavirus particles in feces, rectal swabs, and organ fragments from dogs using transmission electron microscopy techniques.