CHAPTER 1 – The Alimentary System

Vaccine-based prophylaxis has greatly helped to keep distemper disease under control. Notwithstanding, the incidence of canine distemper virus (CDV)–related disease in canine populations throughout the world seems to have increased in the past decades, and several episodes of CDV disease in vaccinated animals have been reported, with nation-wide proportions in some cases. Increasing surveillance should be pivotal to identify new CDV variants and to understand the dynamics of CDV epidemiology. In addition, it is important to evaluate whether the efficacy of the vaccine against these new strains may somehow be affected.

Canine distemper virus.

Measles virus is a negative-sense, single-stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in NTAIL, an α-helical molecular recognition element (α-MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and NTAIL, which shows that most of NTAIL remains disordered in the complex despite P-induced folding within the α-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of NTAIL, and of a bulky globular region, corresponding to XD and to the C terminus of NTAIL (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that NTAIL has an additional site (aa 517–525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded.

Studies on manifestations of canine distemper virus infection in an urban dog population

The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

Identification and characterization of a regulatory domain on the carboxyl terminus of the measles virus nucleocapsid protein.

Twelve gnotobiotic dogs from 2 litters were allotted to 3 groups. Group A dogs received a modified-live polyvalent (canine distemper, adenovirus type 2, and parainfluenza virus and Leptospira -canicola-icterohemorrhagiae bacterin) vaccine 3 days prior to oral inoculation with canine parvovirus (CPV). Group B dogs received CPV alone. Group C dogs received 1 dose of vaccine only. In none of the 9 CPV-inoculated dogs did clinical signs of CPV infection develop, although high serum antibody titers for CPV developed in all of them. However, in 2 of the 5 CPV-inoculated vaccinates, canine distemper virus encephalomyelitis subsequently developed. The results suggested that CPV exerts an immunomodulating effect on canine immune responses and may be responsible for vaccination failures in dogs.

Canine parvovirus infection potentiates canine distemper encephalitis attributable to modified live-virus vaccine.

Summary
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.

J. Rice

Papers

Canine parvovirus infection potentiates canine distemper encephalitis attributable to modified live-virus vaccine.

Isolation and characterization of canine distemper virus nucleocapsid variants.

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

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization