Possible vaccine-induced canine distemper in a South American bush dog (Speothos venaticus).

Suspected vaccine-induced canine distemper was diagnosed in a captive female bush dog (Speothos venaticus). Macroscopic lesions included mild congestion of the gastric mucosa and focal consolidation of the lung. Histopathological lesions included status spongiosis, gliosis, widespread eosinophilic, intranuclear and intracytoplasmic inclusion bodies in neurons, astrocytes and gitter cells of the cerebral, cerebellar and spinal white matter.

Isolation and characterization of canine distemper virus nucleocapsid variants.

Nucleocapsid (NC) variants expressed by the Onderstepoort strain of canine distemper virus (CDV) were ultrastructurally and biochemically characterized. Three isolated variants were defined which corresponded to the three variants observed within the cytoplasm of infected cells. Dense NC (D-NC), isolated on discontinuous caesium chloride (CsCl) isopycnic gradients, had an average density of 1.2971 +/- 0.0042 g/ml. Ultrastructurally, D-NC were 1620.0 +/- 112.1 nm in length with a 20.1 +/- 1.3 nm outer and a 5.8 +/- 0.7 nm inner core diameter. The D-NC protein composition was 89.7% of a 61K protein (N), 8.4% of a 75K protein (P) and 1.9% of a 160K to 200K protein (L). A single species of nucleic acid, 15 kb in length, was isolated from D-NC. Light NC (L-NC), similarly isolated, had an average density of 1.2894 +/- 0.0040 g/ml. L-NC differed ultrastructurally from D-NC in that poor resolution of NC subunits, a larger outer diameter (32.0 +/- 2.8 nm), and a greater inner core diameter (10.4 +/- 0.6 nm) were observed. The average L-NC strand length was 1574.4 +/- 115.8 nm. The protein composition was the same as D-NC with the exception of an additional 70K protein, representing 4.0 to 7.7% of the total L-NC protein mass. A 15 kb nucleic acid was also identified in L-NC, although heightened sensitivity of encapsidated L-NC nucleic acid to non-specific nuclease degradation was observed. The ratio of D-NC to L-NC isolated from individual virus preparations varied and was independent of viral infectivity. A third NC variant, defective-NC (Df-NC), was also identified. This had the lowest density on CsCl gradients (1.2460 +/- 0.0046 g/ml). The Df-NC structures were truncated to a uniform length of 87.0 +/- 5.8 nm. Diameter measurements were between those of D-NC and L-NC, being 24.4 +/- 1.4 nm (outer) and 6.9 +/- 0.4 nm (inner core). Like L-NC, the 70K protein was present but in greater amounts, representing as much as 43.7% of the total Df-NC protein mass. RNase A-sensitive nucleic acid was isolated from Df-NC which ranged in size from 1.16 to 0.67 kb with a majority of the material being 0.86 kb in length. For both L-NC and Df-NC, canine CDV convalescent serum reacted with viral N and P proteins in Western blot analyses but not with the 70K protein, suggesting a host cellular origin for the latter.

Rapid isolation of morbillivirus nucleocapsid for genomic RNA cDNA cloning and the production of specific core protein antisera.

A procedure is described for the rapid isolation of canine distemper virus nucleocapsid, free from contaminating viral non-core and host cellular proteins. Nucleocapsid isolated in this manner is amenable to ultrastructural evaluation, protein isolation for the production of monospecific hyperimmune serum, and genomic RNA isolation for cDNA cloning. Nucleocapsid (NC) and a defective NC variant (Df-NC) isolated from 5.5 x 10(7) Vero cells infected with Ond-CDV is readily visualized on cesium gradients. The calculated density for NC is 1.2976 +/- 0.0033 g/ml and 1.2458 +/- 0.0056 g/ml for Df-NC. Ultrastructurally, NC appears as long uninterrupted strands, 1.6 +/- 0.1 microns in length, 21.2 +/- 1.7 nm in diameter, with well defined capsid subunits. Df-NC are truncated with a uniform length of 85.8 +/- 7.1 nm and a 24.5 +/- 1.3 nm diameter. A total of 2.1 +/- 0.2 mu of NC protein is obtained for every 1 x 10(6) cells infected; 89.7% of this mass is represented by a 61 kDa protein (N), 8.4% by a 75 kDa protein (P), and 1.9% by a 160-200 kDa protein (L), which is in agreement with the NC constituency of other paramyxoviruses. Viral N and P proteins, purified by 7.5% SDS-PAGE, were used in the production of hyperimmune serum. Specificity was demonstrated by Western blot analysis. Both antisera were capable of detecting viral antigen in persistently and lytically CDV infected cells by indirect immunofluorescence. A single high molecular weight species of nucleic acid was isolated from purified nucleocapsids compatible with a 14.6 kb morbillivirus genome. Although the efficiency of RNA extraction from purified NC was low (14.2%), sufficient RNA was obtained for gel analysis and the establishment of genomic RNA cDNA clones.