Detection of Canine Distemper Virus Nucleoprotein RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and Cerebrospinal Fluid from Dogs with Distemper

Abstract

Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.

Virological Survey in free-ranging wildcats (Felis silvestris) and feral domestic cats in Portugal

“…Viral RNA and DNA were simultaneously extracted from stool using the QIAmp 1 Minielute 1 Virus spin Kit (Qiagen, Germany) following the manufacturer instructions. Detection of FCoV RNA was performed according to Herrewegh et al (1995); FPV DNA was screened according to Desario et al (2005) and CDV RNA according to Frisk et al (1999). The amplification products were visualized after electrophoresis in a 2% agarose gel, stained by EtBr in an Image Master 1 VDS (Pharmacia Biotech).…”

Genetic analysis of canine group 2 coronavirus in Korean dogs

“…The canine parainfluenza virus (CPiV), canine herpesvirus (CHV), and canine adenovirus type 2 (CAV-2) were PCR-amplified as previously described (Erles et al, 2004). The canine adenovirus type 1 (CAV-1), canine distemper virus (CDV), and canine influenza virus (CIV) were also amplified using previously published methods (Hu et al, 2001;Frisk et al, 1999;Hoffmann et al, 2001). CRCoV was amplified using the primers described in Table 1.…”

“…Sequence 5 consisted of 30 min at 42 8C, 15 min at 94 8C, 15 min at 94 8C, followed by 35 cycles of 30 s at 95 8C, 30 s at 48-52 8C, and 60 s at 72 8C, and with a final elongation step of 5 min at 72 8C. Amplification of specific genes from other viral strains was carried out according to previously published protocols (Erles et al, 2004;Hu et al, 2001;Frisk et al, 1999;Hoffmann et al, 2001;Ikeda et al, 2000;Pratelli, 2006;Kang et al, 2008;Hozbor et al, 1999). PCR products of the expected sizes were purified by electrophoresis on a 1% agarose gel, followed by extraction using a QIAquick Gel Extraction Kit (QIAGEN, Cat.…”

Genetic diversity of Hungarian canine distemper virus strains

“…CD is caused by the canine distemper virus (CDV) which belongs to the Morbillivirus genus of the Paramyxoviridae virus family. The clinical symptoms of the disease include severe respiratory, digestive and neurological signs with frequently fatal outcome (Appel and Summers, 1995;Stettler et al, 1997;Frisk et al, 1999;Ozkul et al, 2004). The clinical evolution of the infection is characterized by a specific biphasic fever curve, and other clinical signs that do not respond to symptomatic antimicrobial therapy.…”