Development of multiplex RT-PCR and metagenomic analyses of canine infectious respiratory disease complex associated viruses in dogs

Abstract

Canine infectious respiratory disease complex (CIRDC) viruses have been detected in dogs with respiratory illness. The CIRDC is associated with multiple factors depending on host susceptibility, environment and pathogens. Canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAdV-2) and canine herpesvirus 1 (CaHV-1), are all associated with the CIRDC. Environmental factors serve as the one key point for infection. The transmission route can be via community-acquired infection (CAI) or hospital-associated infection (HAI), but the variable factors within these two routes are not well described. Moreover, novel pathogens that could not be identified, were detected from dogs showing respiratory problem. Thus, to allow diagnosis, establish the CIRDC viruses with associated risk factors for infection and discover the other novel pathogen causing respiratory disease in dogs, two conventional multiplex polymerase chain reactions (PCR) were developed to simultaneously identify four RNA and two DNA viruses associated with CIRDC. The developed multiplex PCRs were tested by sensitivity and specificity determination in comparison to conventional simplex PCR and a rapid three-antigen test kit. The two multiplex PCR assays were then validated on 418 respiratory samples collecting from 209 respiratory illness dogs locating in Thailand. In term of analysis of possible risk factors of CIRDC, the sample population of 209 dogs were divided in 133 community acquired infection (CAI) and 76 Hospital associated infection (HAI) groups. Essential signalments, clinical signs, vaccination status, and route of transmission were recorded for further analysis. Either negative-multiplex PCRs or interesting samples were subjected to further investigation by metagenomic analysis using Next Generation Sequencing (NGS) technology. Here, we established the developed multiplex PCR assays, which had a > 87% sensitivity and 100% specificity compared to their simplex counterpart. Compared to the three-antigen test kit, the multiplex PCR assays yielded 100% sensitivity and more than 83% specificity for detection of CAdV-2 and CDV, but not for CIV. Interestingly, all common six viruses were detected in both groups with CIV and CRCoV being predominantly found. Only CDV was significantly more prevalent in CAI than HAI dogs but not for the others. Multiple infections were found in 81.2% and 78.9% of CAI and HAI, respectively. Co-detection of CIV and CRCoV was significantly associated among study groups. Moreover, the clinical severity level was notably related to age of infected dogs, neither vaccination status, sex nor transmission route were noted. Surprisingly, a novel strain of canine bocavirus type 2 (CBoV-2) and canine circovirus (CanineCV) were identified in the samples from dogs that died in Thailand from acute disease, for which no causal etiological pathogen could be identified in routine screening assays. These novel CBoV-2 and CanineCV strains were named as CBOV TH-2016 and CanineCV TH/2016, respectively. Analysis of the complete coding sequences of CBoV TH-2016 and CanineCV TH/2016 showed that these viruses showed evidence of genetic recombination among other published strains by using similarity plot, bootscanning and cocktailed of statistical maximum likelihood algorithms. Use of both conventional or quantitative PCR and in situ hybridization showed the presence of these viruses in several tissues, suggesting hematogenous virus spread. Histopathological lesions associated with these viral infections in several organs and confirming of presence of the CBoV-2 infection using transmission electron microscope provided novel insights in the pathogenicity of canine bocavirus and canine circovirus infections and suggests that a novel recombinant of those viruses, which was discovered from deceased dogs showing negative for routine tests, may result in atypical syndrome.