Effective primary isolation of wild-type canine distemper virus in MDCK, MV1 Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes.
Canine distemper virus (CDV) is an important pathogen of many carnivores. We are developing a field-based model of morbillivirus virulence and pathogenesis through a study of distemper in naturally infected free-ranging raccoons. The isolation of CDV from raccoon tissues is essential for this work. CDV has often been isolated from animals only after co-cultivation of infected tissues with peripheral blood mononuclear cells derived from specific pathogen-free dogs or similar methods. We explored the utility and consequences of a simpler and cheaper alternative: CDV isolation in Vero, MDCK, and MV1 Lu cells. Virus growth was detected first in MDCK cells, whereas viral cytopathic effects were most obvious in Vero cells. CDV growth in MV1 Lu cells was relatively protracted and occurred without the formation of cytopathic effects. In primary CDV isolates, the entire nucleotide sequence of the receptor binding haemagglutinin (H) gene, and subgenomic fusion (F) and phospho (P) protein gene sequences corresponding to nt 5399-5733 and 2132-2563 of CDV reference strain Onderstepoort, respectively, were identical to those in matched infected tissues. Virus isolation confirmed the presence of CDV in instances where RT-PCR failed to detect CDV in infected tissues. Different viral phenotypes and genotypes were detected. The conservation of H gene sequences in primary CDV isolates suggests that MDCK, MV1 Lu, and Vero cells express proper receptors for wild-type CDV.
Lednicky
John A
JA
Department of Pathology, Loyola University Medical Center, 2160 South First Street, Maywood, IL 60153, USA. jlednic@lumc.edu
Meehan
Thomas P
TP
Kinsel
Michael J
MJ
Dubach
Jean
J
Hungerford
Laura L
LL
Sarich
Nicolene A
NA
Witecki
Kelley E
KE
Braid
Michael D
MD
Pedrak
Casandra
C
Houde
Christiane M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004216:Distemper / Q000821:virology*; D004217:Distemper Virus, Canine / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D004285:Dogs; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011821:Raccoons / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006389:Hemagglutinins, Viral; D010750:Phosphoproteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; C076619:F protein, Phocine distemper virus