RESUMO
The nucleocapsid (N) protein of all morbilliviruses has a highly conserved central region that is thought to interact with and encapsidate the viral RNA. The C-terminal third of the N protein is highly variable among morbilliviruses and is thought to be located on the outer surface and to be available to interact with other viral proteins such as the phosphoprotein, the polymerase protein and the matrix protein. Using reverse genetics, a chimeric rinderpest virus (RPV)/peste-des-petits-ruminants virus (PPRV) was rescued in which the RPV N gene open reading frame had been replaced with that of PPRV (RPV-PPRN). The chimeric virus maintained efficient replication in cell culture. Cattle vaccinated with this chimeric vaccine showed no adverse reaction and were protected from subsequent challenge with wild-type RPV, indicating it to be a safe and efficacious vaccine. The carboxyl-terminal variable region of the rinderpest N protein was cloned and expressed in Escherichia coli. The expressed protein was used to develop an indirect ELISA that could clearly differentiate between RPV- and PPRV-infected animals. The possibility of using this virus as a marker vaccine in association with a new diagnostic ELISA in the rinderpest eradication programme is discussed.
Assuntos
Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/imunologia , Vírus da Peste Bovina/genética , Vírus da Peste Bovina/imunologia , Vacinas Virais/genética , Animais , Anticorpos Antivirais/análise , Sequência de Bases , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Quimera/genética , Quimera/imunologia , Chlorocebus aethiops , Clonagem Molecular , DNA Viral/genética , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Genes Virais , Marcadores Genéticos , Masculino , Peste dos Pequenos Ruminantes/diagnóstico , Peste dos Pequenos Ruminantes/imunologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Recombinação Genética , Peste Bovina/diagnóstico , Peste Bovina/imunologia , Peste Bovina/prevenção & controle , Células Vero , Vacinas Virais/imunologia , Cultura de VírusRESUMO
A specific interaction between the F and H proteins is required to enable fusion of the virus and host cell membranes and in some cases these proteins are not interchangeable between related viruses of the family Paramyxoviridae. For example, the F and H proteins of two ruminant morbilliviruses, rinderpest virus (RPV) and Peste-des-petits-ruminants virus (PPRV), are not interchangeable since viable virus could not be rescued from cDNA constructs where an individual glycoprotein gene of RPV was replaced with that from PPRV. To investigate which domain of the H protein, extracellular or cytoplasmic/transmembrane, was most important for preventing this interaction, two chimeric H gene constructs were made where the normal H gene of RPV was substituted with variant H genes where the transmembrane/cytoplasmic tail region (pRPV2C-PPRTm) or the whole ectodomain (pRPV2C-PPRExt) were derived from PPRV. Chimeric viruses were rescued from both the constructs and, while RPV2C-PPRTm virus grew to as high titres as the parent virus, RPV2C-PPRExt virus was extremely debilitated with respect to growth in tissue culture. Thus the ectodomain of H is the most important region required for effective interactions of the two glycoproteins for the recovery of viable virus. Nevertheless, the transmembrane/cytoplasmic domain of RPV alone can allow a chimeric virus to be rescued, which was not possible when the complete H gene was derived from PPRV. Both versions of the H protein and also the F protein were found to be incorporated into the envelope of the budded virions.
Assuntos
Hemaglutininas Virais/química , Hemaglutininas Virais/fisiologia , Vírus da Peste Bovina/fisiologia , Replicação Viral/genética , Animais , Chlorocebus aethiops , Citoplasma , DNA Complementar , Expressão Gênica , Glicoproteínas/metabolismo , Hemaglutininas Virais/genética , Proteínas de Membrana , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/fisiologia , Estrutura Terciária de Proteína/genética , RNA Mensageiro/análise , RNA Viral/análise , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Peste Bovina/genética , Células Vero , Proteínas Virais de Fusão/metabolismo , Ensaio de Placa Viral , Proteínas Virais/análiseRESUMO
The gene encoding the phosphoprotein of the vaccine strain of Peste des petits ruminants (PPR) virus (Nigeria 75/1 vaccine strain) has been cloned and its nucleotide sequence been determined. This gene is 1655 nucleotides long and encodes two overlapping open reading frames (ORFs). Translation from the first AUG would produce a polypeptide of 509 amino acid residues with a predicted molecular mass of 54.9 kDa, the longest of the published morbillivirus P proteins. Translation from the second AUG would produce a protein of 177 amino acid residues with a predicted molecular mass of 20.3 kDa, analogous to the C proteins of other morbilliviruses. Evidence was found for the production of two types of P mRNA transcript, one a faithful transcript of the gene and the other with an extra G residue inserted at position 751. Translation from the first AUG of this second mRNA would produce a protein of 298 amino acids, with a predicted molecular mass 32.3 kDa, analogous to the V protein produced by other morbilliviruses. Sequences of the predicted P, C and V proteins were compared with those of the other morbillivirus sequences available to date. The P protein was found to be the most poorly conserved of the morbillivirus proteins, the amino acid identity ranging from 54% in case of Canine distemper virus (CDV) to 60% in the case of the Dolphin morbillivirus (DMV).
