Animal and ethics
A total of 190 dogs, comprising both healthy dogs (n = 76) and dogs with respiratory symptoms (n = 114), were included in this study. The research assessed nasal swab (NS) and oropharyngeal swab (OS) collected between January and November 2020. The samples from the healthy group were collected from clinically healthy dogs during routine dental scaling at the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand. Samples from dogs with respiratory signs were collected from owned dogs that visited the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University, Bangkok and vicinities, Thailand. Clinical signs of dogs included sneezing, coughing, nasal discharge, and bronchopneumonia which was diagnosed based on thoracic radiography. Dogs showing respiratory signs caused by a nasal foreign body, heart disease, or functional tracheal disease; dogs exhibiting enteric diseases; and dogs that received a vaccination within the 4 weeks prior to sampling were excluded.
Essential signalments including sex, breed, sterilization status, and respiratory clinical signs were recorded for further analysis. Dogs’ age was classified as followed: junior dogs (age < 1.5 years), adult dogs (age ≥ 1.5 to < 6 years), and senior dogs (age ≥ 6 years)42,43. To support the possible potential role of CanineCV in respiratory disease, dogs that had died from bronchopneumonia submitting for routine necropsy at Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, were included in the study. This research was approved by the Institutional Animal Care and Use Committee (IACUC) (No. 2031014) and the Institutional Biosafety Committee (IBC) (No. 1931036) of Chulalongkorn University. This study is reported in accordance with ARRIVE guidelines.
Sample collection and DNA extraction
For the NS sampling site, the swabs (Puritan, Guilford, USA) were inserted into the nostril to about 3 cm or shallower according to the dog breed, whereas OSs were placed on the oropharynx around the tonsils and then rolled gently. NSs and OSs collected from all dogs were immersed in 1% (v/v) sterile phosphate buffer saline (PBS) and then subjected to genomic extraction immediately using the Viral DNA/RNA extraction kit (Geneaid, Ltd., Taipei, Taiwan) following the manufacturer’s protocol. The extracted nucleic acids were quantified and qualified on the basis of a 260/280 absorbance ratio using a Nanodrop® Lite spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The extracted samples were kept at − 80 °C until used.
A total of 15 lungs from necropsied dogs suffering from bronchopneumonia were obtained and divided for molecular assays and pathological examination. For molecular detection, the frozen lung tissues were minced, and homogenized in 1% PBS by a tissue homogenizer and clarified by centrifugation at 10,000×g for 3 min. The supernatants were collected for genomic extraction as mentioned earlier and stored at − 80 °C until assayed. The remaining part of the lungs was fixed in 10% neutral buffered formalin for pathological purposes. Lung tissues from necropsied dogs that died from non-respiratory disease were included for CanineCV detection by cPCR, comprising dogs suffering from cardiovascular failure (n = 13) and accidentally traumatic injury (n = 15).
Virus-associated Canine Infectious Respiratory Disease Complex (CIRDC) detection
Common canine respiratory viruses, including canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 1 and 2 (CAdV-1 & 2), and canine herpesvirus 1 (CaHV-1), were screened in the samples (NS and OS) from both healthy and respiratory dogs, as well as homogenized lung tissues, using multiplex reverse-transcription (RT)-PCR/PCR assays as previously described44. The information regarding the virus-associated CIRDC tests was used for further interpretation.
CanineCV detection and full-length genome amplification
The nucleic acids extracted from both studied groups were tested for CanineCV genome by conventional PCR (cPCR) using Gotaq Green Master mix (Promega, WI, USA) and specific primers (F605-R1041 and F1022-R1538), to amplify the partial Rep and Cap genes (Table 4). The thermocycling condition consisted of an initial denaturation at 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and then a final extension at 72 °C for 7 min. To complete the full-length genome of CanineCV, multiple primer pairs (F1349 or F1372-R110 and F2014-R764) were used to amplify the CanineCV sequences (Table 4). The thermocycling condition was set as the same condition described above, except for the annealing temperature, which was changed to 58 °C for 30 s with an extension time at 72 °C for 45 s. The positive control for CanineCV cPCR was retrieved from a previous publication27. A no-template control (NTC) was used as the negative control. The PCR products were separated on 1.5% (w/v) agarose gel containing ethidium bromide and were visualized under a UV transilluminator.
To confirm the CanineCV nucleotide sequences, the PCR products were purified using NucleoSpin Extract II (Macherey–Nagel, Düren, Germany) and submitted for bidirectional Sanger sequencing (Macrogen Inc., Seoul, South Korea). The obtained sequences were initially analyzed by alignment with previously published CanineCV genomes available in the GenBank database using BLASTn analysis. For full-length genome characterization, several genome products from multiple cPCRs with specific primer pairs were visualized, purified, and submitted for Sanger sequencing as described above. The derived genetic sequences were initially blasted with the previous CanineCV sequences deposited in GenBank to confirm the presence of CanineCV genomes. Subsequently, the derived genetic sequences were aligned and assembled using BioEdit software package version 7.2 with the ClustalW function.
Presence of CanineCV in tissues by qPCR
To quantify the viral load in different tissues (Supplementary Table S6) of CanineCV positive carcasses, SYBR Green-based qPCR was performed using the KAPA SYBR® Fast qPCR Master Mix (2X) Universal kit (KAPABIOSYSTEMS, Sigma-Aldrich, Cape Town, South Africa). The reaction consisted of a 250 nM final concentration of each primer (F247–R502) (Table 4), which were used to amplify the partial Rep gene, giving a product size of 254 bp. The amplification was processed in a Rotor-Gene Q real-time PCR cycler (Qiagen GmbH, Hilden, Germany) with the protocol of initial denaturation at 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s to acquire the fluorescence A green. Then, melt curve analysis was performed to define the melting temperature of the amplicon. A positive result was expected to melt at 85.0 °C to 85.5 °C when the temperature was increased from 60 to 95 °C. The software reported the cycling A green and melt A green compared with the NTC and positive control retrieved from the positive clinical samples confirmed by sequencing. An output Ct value was used to estimate the viral copy number in each organ.
Phylogenetic and recombination analyses
The phylogenetic tree was constructed using MEGA software package version 7.0. The maximum likelihood (ML) method, based on the general time reversible model (GTR) with a gamma distribution and invariable sites (G + I) together with 1,000 bootstrap replicates, was used to evaluate the relationship between these obtained CanineCV strains and the other strains deposited in the GenBank database. Additionally, the CanineCV alignment was subjected to recombination analysis using the Recombination Detection Program software package version 4.0 (RDP4). The RDP included seven recombination detection methods (RDP, GeneConv, BootScan, MaxChi, Chimera, SiScan, and 3Seq) to analyze the recombination breakpoint. A potential recombination sequence was accepted with a p-value of ≤ 10–10 from at least four out of the seven methods. Subsequently, the potential recombination strains derived from the RDP analysis were subjected to similarity plot and Bootscan analysis using SIMPLOT software package v. Beta 4.94 to additionally confirm the presence of the recombination breakpoint. The program was set and analyzed according to previous publications27.
Pathological examination
After fixing the fresh lung tissues in 10% neutral buffered formalin for 48 h, tissues were further histologically processed and embedded in paraffin wax, sectioned at a 4 µm-thickness, and stained with Hematoxylin and Eosin (HE). Microscopic findings were inspected by Thai board-certified veterinary pathologist (ST).
In situ hybridization (ISH)
The CanineCV-PCR positive lungs were further investigated for the CanineCV genome in the tissues by ISH. The digoxigenin (DIG)-labeled probe covering 437 bp of the partial CanineCV Rep gene was constructed by utilizing the PCR DIG probe synthesis kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. Briefly, the CanineCV-DIG probe was synthesized with the same thermal cycling conditions described in cPCR for the partial Rep gene. The DIG-labeled oligonucleotides were used instead of the normal oligonucleotides. Then, the constructed CanineCV-DIG probe was observed by visualizing the product size on 1.5% (w/v) agarose gel electrophoresis.
After sections were deparaffinized and rehydrated, they were then pretreated with 0.2 N hydrochloric acid (HCl) at room temperature for 20 min, followed by citrate buffer (pH 6) at 95 °C for 20 min. The slides were incubated with 10 ng/mL proteinase K (VWR, Radnor, PA, USA) in 1X Tris-NaCl-EDTA (TNE) buffer at 37 °C for 20 min and soaked with 0.4% cold formaldehyde solution. Then, slides were prehybridized with prehybridization buffer containing 50% (v/v) formamide in 4X saline-sodium citrate (SSC) buffer at 37 °C for at least 15 min and subsequently incubated with a hybridization buffer containing 20X SSC, 5X Denhardt’s solution, 100 ug/mL salmon sperm DNA, 0.5% (w/v) sodium dodecyl sulfate, and 10 ng of CanineCV-DIG-labeled probe per slide at 50 °C overnight in the slide incubator. After incubation, the slides were stringently washed in a series of 2X SSC at 37 °C, 1X SSC at 42 °C, and 0.5X SSC at 42 °C, each for 15 min, respectively. After that, the slides were then incubated in 5% bovine serum albumin (BSA) for non-specific binding blocking at room temperature for 30 min. The hybridization reactions were visualized by incubating with 1:200 anti-DIG-AP Fab fragments (Roche, Mannheim, Germany) in 1X blocking solution, and the chromogenic signals were developed using PermaRed/AP (Diagnostic BioSystems, CA, USA) applied in a dark chamber at room temperature for 40 min. Slides were then counterstained with hematoxylin. The dark red dots presented within the cellular structure were considered as positive results, whereas the CanineCV PCR-negative section and unrelated probe, canine bocavirus (CBoV) probe45, were used as negative controls.
Statistical analysis
The associations between the presence of CanineCV and other variable data, including age, sex, breed, sterilization status, sampling route, co-detection with CIRDC viruses, and clinical symptoms of sampled dogs, were calculated using Pearson’s chi-squared test or Fisher’s exact test depending on the population size being assessed in each factor. The output data were considered statistically significant at a p-value < 0.05. The odds ratio was then calculated to measure the strength of association between each factor and CanineCV occurrence. The statistical analyses were conducted using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA).
Ethics statement
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received by the Chulalongkorn University Animal Care and Use Committee (No. 2031014). All procedures were done in accordance with the relevant guidelines and regulations. Authors confirm that this study is reported in accordance with ARRIVE guidelines.
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