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.

FIG. 1

Schematic drawing of the CDV genome and mRNA with location of the primers used for PCR. P/V/C, phosphoprotein; M, matrix protein; F, fusion protein; H, hemagglutinin; L, large protein; nt, nucleotide. Arrows indicate directions of primers. Numbers are molecular sizes (in base pairs). Moderate, high, and little or no, sequence homology of the NP gene within the genus morbillivirus.

FIG. 2

Cervical spinal cord of dog 38 showing chronic myelitis with malacia, demyelination, and moderate perivascular lymphohistiocytic cuffs. Hematoxylin and eosin stain was used. Magnification, ×35.

FIG. 3

Cerebellum of dog 32 showing a strong positive signal for NP antigen in endothelial cells and intravascular lymphocytes interpreted as ongoing viremia. The avidin-biotin complex method was used. Magnification, ×350.

FIG. 4

RT-PCR results (top panels) obtained with PP-I from CDV laboratory strains, CDV field isolates, Edmonston strain of measles virus, porpoise morbillivirus, and canine parainfluenza virus RNA and results obtained by Southern blot analysis (bottom panels). (A) Lanes 1, CDV-Convac; lanes 3, canine parainfluenza virus type 2; lanes 5, porpoise morbillivirus; and lanes 7, R252-CDV. (B) Lanes 1, Ond-CDV; lanes 3, Edmonston strain of measles virus; lanes 5, field isolate 98/91; lanes 7, field isolate 2582/90. Lanes with even numbers, negative controls (noninfected Vero cells); lanes M₁, DIG-labeled molecular size marker; lane M₂, molecular size marker (100-bp ladder). Numbers on the left and right are molecular sizes (in base pairs).

FIG. 5

RT-PCR results (top panels) with PP-III from CDV laboratory strains, CDV field isolates, the Edmonston strain of the measles virus, porpoise morbillivirus, and canine parainfluenza virus RNAs and results obtained by Southern blotting analysis (bottom panels). (A) Lanes 1, CDV Convac; lanes 3, canine parainfluenza virus type 2; lanes 5, porpoise morbillivirus; and lanes 7, CDV R252. (B) Lanes 1, Ond-CDV; lanes 3, Edmonston strain of measles virus; lanes 5, field isolate 98/91; and lanes 7, field isolate 2582/90. Lanes with even numbers, negative controls (noninfected Vero cells); lanes M₁, DIG-labeled molecular size marker; lanes M₂, molecular size marker (100-bp ladder). Numbers on the left and right are molecular sizes (in base pairs).

FIG. 6

RT-PCR amplification of CDV NP nucleic acid (A) and Southern blot analysis (B) of CSF, serum, and whole blood from dog 32 with PP-I. Lanes 1, 3, and 5, amplicons in CSF, serum, and whole blood, respectively; lanes 2, 4, and 6, controls (noninfected Vero cells); lanes M₁, DIG-labeled molecular size marker; lanes M₂, molecular size marker (100-bp ladder). Numbers on the left and right are molecular size markers.

FIG. 7

Alignment of the nucleotide sequence of the NP gene of different distemper virus strains (five CDV isolates and the Rockborn strain) compared to that of the NP gene of Ond-CDV GenBank-EMBL data bank accession numbers are as follows: strain 13, AF166268 (isolate 833-Gi95); strain 23, AF166269 (isolate 1127-Gi95); strain 29, AF166270 (isolate 2852-Gi95); strain 30, AF166271 (isolate 2153-Gi95); strain 40, AF166272 (isolate 2495-Gi95); CDV Rockborn (CDV-ROCK), AF166273.