Materials and Methods
In July of 2015, a six-month-old sick guiña was recovered in an urban
area from Curicó (Chile) by the Agricultural and Livestock Service (SAG)
of Chile. The animal was submitted to the Center for Rehabilitation of
Wild Fauna of the Universidad de Concepción (Chillán, Chile). The
individual presented an intermittent hemorrhagic diarrhea, depression
and anorexia during the clinical exam. A blood sample was collected to
identify a possible infectious cause. Diagnostic test against FelV, FIV
by Nested PCR (Mora et al., 2015) and against CPV by PCR were
performed, resulting positive for CPV and negative to FeLV and FIV. A
few weeks later, the animal died, and necropsy was carried out. Stool,
stomach, small intestine and spleen tissues were collected.
DNA was extracted from blood sample using ThermoFisher Scientific DNA®
Extraction from Blood (processed at the Virology Laboratory, Faculty of
Veterinary Sciences, University of Concepción (Chillán, Chile)).
DNA
was extracted from tissue samples using DNeasy Blood and Tissue Kit and
the QIAamp DNA Stool Mini Kit; Qiagen (Processed at the Molecular
Biology Laboratory, Faculty of Sciences, University of Chile (Santiago,
Chile)). The extracted genomic DNAs were stored at -20°C.
For CPV diagnosis, a small viral fragment of VP2 (583 bp) was amplified
following Buonavoglia et al ., (2001). Furthermore, seven samples
from Chilean domestic dogs were selected for complete coding genome
sequencing. These samples were previously characterized by Castilloet al ., (2020) using sequencing of the VP2 gene. Therefore, the
complete coding genome (4269 pb) from dogs and guiña were amplified
following Pérez et al ., (2014).
The dataset consisted of sequences generated in this study and CPV
complete coding genome sequences retrieved from the GenBank database.
The filter criterion was to select the sequences according to its
subtype, country and year of collection and host species other than dog.
The sequences were aligned using MUSCLE v3.8.3 (Edgar, 2004)
incorporated in AliView v1.26 (Larsson, 2014). The best-fit evolutionary
model of nucleotide substitution was selected according to the Akaike
Information Criterion (AIC) in jModelTest v2.1.6 software (Posada,
2008). Phylogenetic tree was inferred using the maximum likelihood
method with RaxML-HPC2 v8.2.12 (Stamatakis, 2014) available in CIPRES
Science Gateway (Miller et al ., 2010). The statistical support of
the nodes was evaluated using 1000 bootstrap replicates.