Development of a Challenge-Protective Vaccine Concept by Modification of the Viral RNA-Dependent RNA Polymerase of Canine Distemper Virus

ABSTRACT We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a “quick response” in vaccine development against well-known and “new” viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

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Molecular detection and phylogenetic relationship of wild-type strains of canine distemper virus in symptomatic dogs from Uberlândia, Minas Gerais

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-7052 ◽

2015 ◽

Vol 67 (6) ◽

pp. 1510-1518

Author(s):

S.A. Headley ◽

T.R. Santos ◽

L. Bodnar ◽

J.P.E. Saut ◽

A.P. Silva ◽

...

Keyword(s):

Canine Distemper Virus ◽

Phylogenetic Analyses ◽

Clinical Manifestations ◽

N Gene ◽

Clinical Samples ◽

Canine Distemper ◽

Wild Type ◽

Age Related ◽

Relationship Of ◽

Type Strains

This study investigated the occurrence of canine distemper virus (CDV) by evaluating the presence of viral RNA within urine samples of dogs from Uberlândia, MG, with clinical manifestations suggestive of infection by CDV by targeting the CDV N gene. Of the clinical samples collected ( n =33), CDV viruria was detected in 45.5%. Five dogs died spontaneously; all had characteristic CDV-associated histopathological alterations and demonstrated CDV viruria. Statistical analyses revealed that the age, gender, breed, or the organ system of the dog affected had no influence on the occurrence of canine distemper. Myoclonus and motor incoordination were the most significant neurological manifestations observed. A direct association was observed between keratoconjunctivitis and dogs with CDV viruria. These findings suggest that CDV viruria in symptomatic dogs might not be age related, and that symptomatic dogs can demonstrate clinical manifestations attributed to CDV without viruria identified by RT-PCR. Additionally, the results of the sequence identities analysed have suggested that all Brazilian wild-type strains of CDV currently identified are closely related and probably originated from the same lineage of CDV. Nevertheless, phylogenetic analyses suggest that there are different clusters of wild-type strains of CDV circulating within urban canine populations in Brazil.

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Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

Journal of Virology ◽

10.1128/jvi.73.3.2263-2269.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2263-2269 ◽

Cited By ~ 31

Author(s):

Pascal Cherpillod ◽

Karin Beck ◽

Andreas Zurbriggen ◽

Riccardo Wittek

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Translation Initiation ◽

Fusion Proteins ◽

Canine Distemper Virus ◽

Protein A ◽

Biological Properties ◽

Canine Distemper ◽

Wild Type ◽

Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

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Disease Duration Determines Canine Distemper Virus Neurovirulence

Journal of Virology ◽

10.1128/jvi.00818-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 12066-12070 ◽

Cited By ~ 16

Author(s):

François Bonami ◽

Penny A. Rudd ◽

Veronika von Messling

Keyword(s):

Canine Distemper Virus ◽

Disease Duration ◽

Subsequent Development ◽

Neurological Involvement ◽

Disease Course ◽

Canine Distemper ◽

Wild Type ◽

Neurological Signs ◽

H Protein ◽

Type Strains

ABSTRACT The Morbillivirus hemagglutinin (H) protein mediates attachment to the target cell. To evaluate its contribution to canine distemper virus neurovirulence, we exchanged the H proteins of the wild-type strains 5804P and A75 and assessed the pathogenesis of the chimeric viruses in ferrets. Both strains are lethal to ferrets; however, 5804P causes a 2-week disease without neurological signs, whereas A75 is associated with a longer disease course and neurological involvement. We observed that both H proteins supported neuroinvasion and the subsequent development of clinical neurological signs if given enough time, demonstrating that disease duration is the main neurovirulence determinant.

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Probing Morbillivirus Antisera Neutralization Using Functional Chimerism between Measles Virus and Canine Distemper Virus Envelope Glycoproteins

Viruses ◽

10.3390/v11080688 ◽

2019 ◽

Vol 11 (8) ◽

pp. 688 ◽

Cited By ~ 7

Author(s):

Miguel Angel Muñoz-Alía ◽

Stephen J. Russell

Keyword(s):

Neutralizing Antibodies ◽

Measles Virus ◽

Canine Distemper Virus ◽

Reverse Genetics ◽

Canine Distemper ◽

Virus Envelope ◽

Live Virus ◽

Virus Challenge ◽

Virus Attachment ◽

Globular Head

Measles virus (MeV) is monotypic. Live virus challenge provokes a broadly protective humoral immune response that neutralizes all known measles genotypes. The two surface glycoproteins, H and F, mediate virus attachment and entry, respectively, and neutralizing antibodies to H are considered the main correlate of protection. Herein, we made improvements to the MeV reverse genetics system and generated a panel of recombinant MeVs in which the globular head domain or stalk region of the H glycoprotein or the entire F protein, or both, were substituted with the corresponding protein domains from canine distemper virus (CDV), a closely related morbillivirus that resists neutralization by measles-immune sera. The viruses were tested for sensitivity to human or guinea pig neutralizing anti-MeV antisera and to ferret anti-CDV antisera. Virus neutralization was mediated by antibodies to both H and F proteins, with H being immunodominant in the case of MeV and F being so in the case of CDV. Additionally, the globular head domains of both MeV and CDV H proteins were immunodominant over their stalk regions. These data shed further light on the factors constraining the evolution of new morbillivirus serotypes.

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Prevalence of canine distemper virus in wild mustelids in the Czech Republic and a case of canine distemper in young stone martens

Veterinární Medicína ◽

10.17221/2057-vetmed ◽

2008 ◽

Vol 52 (No. 2) ◽

pp. 69-73 ◽

Cited By ~ 5

Author(s):

L. Pavlacik ◽

V. Celer ◽

P. Koubek ◽

I. Literak

Keyword(s):

Czech Republic ◽

Neutralizing Antibodies ◽

Canine Distemper Virus ◽

Meles Meles ◽

Acute Stage ◽

Direct Immunofluorescence ◽

Canine Distemper ◽

The Czech Republic ◽

Mustela Nivalis ◽

Pine Martens

Between 2001 and 2003, a total of 194 samples of brain tissues of wild mustelids from the Czech Republic were tested for the presence of canine distemper virus (CDV) by direct immunofluorescence examination. Out of 21 animals exhibiting symptoms of the disease or changed behaviour, one mustelid was CDV positive (5% prevalence). In this group, 1 out of 18 stone martens (Martes foina) was CDV positive, while 2 pine martens (Martes martes) and 1 Eurasian badger (Meles meles) were CDV negative. Of 173 animals with unknown case history, 1 sample was positive (0.6% prevalence). In this group of animals, 1 out of 19 Eurasian badgers was positive, and stone martens (n = 96), pine martens (n = 4), polecats (Mustela putorius) ((n = 28), steppe polecats (Mustela eversmani) (n = 4), common weasels (Mustela nivalis) (n = 4), stoats (Mustela erminea) (n = 3) and American minks (Mustela vison) (n = 19) were negative. Clinical distemper was demonstrated in three stone marten pup siblings. In two of the siblings, CDV was demonstrated in footpads. The third of the siblings survived the acute stage of the disease and had virus neutralizing antibodies from the end of the acute stage until 6 months after the end of the acute stage, with a maximum antibody titre of 32. During the acute stage and 7 months after the end of the acute stage, no virus neutralizing antibodies were found.

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Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.404 ◽

1986 ◽

Vol 6 (2) ◽

pp. 404-410 ◽

Cited By ~ 6

Author(s):

T Fujimura ◽

R B Wickner

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Polymerase Activity ◽

Nonpermissive Temperature ◽

Wild Type ◽

Rna Dependent Rna Polymerase ◽

Virus Like Particles ◽

Rna Polymerase Activity ◽

Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

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Canine Distemper Virus Uses both the Anterograde and the Hematogenous Pathway for Neuroinvasion

Journal of Virology ◽

10.1128/jvi.01034-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9361-9370 ◽

Cited By ~ 74

Author(s):

Penny A. Rudd ◽

Roberto Cattaneo ◽

Veronika von Messling

Keyword(s):

Measles Virus ◽

Canine Distemper Virus ◽

Fluorescent Protein ◽

Early Time ◽

Olfactory Nerve ◽

The Body ◽

Target Cells ◽

Canine Distemper ◽

Invasion Routes ◽

Green Fluorescent

ABSTRACT Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.

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Efficient Interaction between Arenavirus Nucleoprotein (NP) and RNA-Dependent RNA Polymerase (L) Is Mediated by the Virus Nucleocapsid (NP-RNA) Template

Journal of Virology ◽

10.1128/jvi.00103-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5734-5738 ◽

Cited By ~ 3

Author(s):

Masaharu Iwasaki ◽

Nhi Ngo ◽

Beatrice Cubitt ◽

Juan C. de la Torre

Keyword(s):

Rna Polymerase ◽

Gene Transcription ◽

Viral Genome ◽

Rna Replication ◽

Rna Dependent Rna Polymerase ◽

Rna Sequences ◽

L Protein ◽

Virus Rna

In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the twotrans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5′ and 3′ termini of the viral genome.

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