ABSTRACT
Marek's disease virus (MDV) GX0101, which is a field strain with a naturally occurring insertion of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) fragment, shows distinct biological activities. Deletion of the meq gene in GX0101 contributes to its complete loss of pathogenicity and oncogenicity in SPF chickens, but this virus has a kanamycin resistance gene (kan(r)) residual at the site of meq gene. In the present study, the kan(r) was knocked out and a meq-null virus with a good replicative ability termed SC9-1 was selected. In vivo studies showed that SC9-1 had no pathogenicity or tumorigenicity to chickens. There were no obvious impacts on chicken weight, immune organ index or antibody levels induced by avian influenza virus (AIV)/newcastle disease virus (NDV) inactivated vaccines compared with the control group. The SC9-1 virus provided superior protection than CVI988/Rispens vaccine in both SPF chickens and Hy-line brown chickens when challenged with a very virulent MDV (rMd5 strain). There was no obvious change in SC9-1 protection against MDV rMd5 in SPF chickens after 20 passages in chicken embryonic fibroblast cells (CEFs). In conclusion, SC9-1 is a safe and effective vaccine candidate for the prevention of Marek's disease.
Subject(s)
Mardivirus/genetics , Mardivirus/immunology , Marek Disease Vaccines/immunology , Marek Disease/prevention & control , Reticuloendotheliosis virus/genetics , Animals , Cells, Cultured , Chickens , Fibroblasts/virology , Gene Deletion , Genes, Viral , Genomic Instability , Marek Disease Vaccines/administration & dosage , Marek Disease Vaccines/genetics , Marek Disease Vaccines/isolation & purification , Recombination, Genetic , Terminal Repeat Sequences , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/isolation & purification , Virus CultivationABSTRACT
The complete DNA sequence of Marek's disease virus (MDV) serotype 1 vaccine strain 814 was determined. It consisted of 172,541 bp, with an overall gene organization identical to that of the MDV-1 type strains. Comparative genomic analysis of vaccine strains (814 and CVI988) and other strains (CU-2, Md5, and Md11) showed that 814 was most similar to CVI988. Several unique insertions, deletions, and substitutions were identified in strain 814. Of note, a 177-bp insertion in the overlapping genes encoding the Meq, RLORF6, and 23-kDa proteins of strain 814 was identified, and a 69-bp deletion was also located in the origin of replication site (Ori) in the gene encoding RLORF12. Compared to the CVI988 vaccine strain, a deletion of 510 bp was identified in the UL36 gene. These analyses identified key mutations in the 814 strain and the vaccine strain that could be exploited for future MDV vaccine design.
Subject(s)
Herpesvirus 2, Gallid/genetics , Marek Disease Vaccines/genetics , Marek Disease/virology , Poultry Diseases/virology , Amino Acid Sequence , Animals , Base Sequence , Chickens , DNA, Viral/genetics , Genome, Viral , Genomics , Herpesvirus 2, Gallid/classification , Marek Disease Vaccines/classification , Marek Disease Vaccines/isolation & purification , Molecular Sequence Data , Mutation , Open Reading Frames , Phylogeny , Sequence Homology, Amino AcidABSTRACT
Marek's disease virus (MDV), an oncogenic alphaherpesvirus, induces a rapid onset T-cell lymphoma and demyelinating disease in chickens. Since the 1970s the disease has been controlled through mass vaccination with herpesvirus of turkeys [meleagrid herpesvirus type 1 (MeHV-1)]. Over time this vaccine's efficacy decreased, and in the 1980s a bivalent vaccine consisting of MeHV-1 and a non-oncogenic gallid herpesvirus type 3 (GaHV-3) strain known as SB-1 was introduced. The complete DNA sequence (165,994 bp) of this GaHV-3 strain was determined using 454 pyrosequencing. A total of 524 open reading frames (ORFs) were examined for homology to protein sequences present in GenBank using BLAST (E-values <0.9). Of the 128 ORF hits, 75 ORFs showed homology to well-characterized alphaherpesviral proteins. Phylogenetically, this strain partitions in its own branch along with the GaHV-3 strain HPRS24 and shows more relatedness to MeHV-1 than gallid herpesvirus type 2 (GaHV-2, Marek's disease virus). When comparing the GaHV-3 ORFs to their homologues in MeHV-1 and GaHV-2, a greater percentage of amino acid similarity was found with homologous ORFs in the genome of SB-1 than with those in the HPRS24 genome. Overall, twice as many of the 75 ORFs within the SB-1 genome showed greater sequence identities and similarities to homologous ORFs in the Marek's disease genome than those within the HPRS24 genome. This paper describes the sequence difference between the two GaHV-3 genomes. Overall 19 ORFs differ in the number of predicted amino acids; of these, eight (U(L)3.5, U(L)5, U(L)9, U(L)28, U(L)30, U(L)36, U(L)37, and U(L)50) encode well-characterized alphaherpesviral proteins A sequence within the unique short region of the SB-1 genome exhibited significant sequence homology to long terminal repeat (LTR) sequences of avian retroviruses. This sequence was only found in the SB-1 genome and not the HPRS24 genome.
Subject(s)
Herpesvirus 2, Gallid/genetics , Marek Disease Vaccines/genetics , Marek Disease/virology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chickens , Genome, Viral , Herpesvirus 2, Gallid/chemistry , Herpesvirus 2, Gallid/classification , Herpesvirus 2, Gallid/isolation & purification , Marek Disease Vaccines/chemistry , Marek Disease Vaccines/classification , Marek Disease Vaccines/isolation & purification , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Alignment , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The study describes three polymerase chain reaction (PCR) systems for the CVI988 vaccine virus: the meq gene, the MDV BamHI-D/H 132 bp tandem repeat fragment and the MDV-gB gene. Whereas the PCR product of virulent MDV strains and of the CVI988 virus strain with the meq and the 132 bp primer sets differed for the two templates, the MDV-gB PCR products were similar. The sensitivity of the three PCRs was determined for the two templates: the CVI988 DNA was detected up to 2.48 plaque forming units, and a MDV-1 DNA, was amplified with the 132 bp primers up to the 10(-3) DNA dilution, and up to the 10(-2) with the MDV-gB and meq gene primers. As conventional detection for the CVI988 vaccine virus is by tissue culture, the aim was to analyse the feasibility of the molecular detection of the vaccine virus in the vaccinated chick. In two experimental trials employing specific pathogen free and commercial Lohmann chicks, respectively, the vaccine virus replicated to a limited extent; it was detected only in the spleen of up to 60% chicks at 2-4 weeks and in one chick at 3 weeks, respectively. The survey of three commercial Lohmann flocks, kept in biosecurity conditions, revealed the vaccine virus only in the spleen of 40% of 30-day-old chicks. The present study shows that CV1988 DNA is present in vaccinated chicks in a low quantity and it is difficult to detect directly from the chick, probably because vaccine viruses are latent in vivo. For an efficient detection it is pertinent to cultivate the vaccine virus on chicken embryo fibroblasts (CEF), as then the virus escapes the latent state, enters into the productive mode of replication, and a high viral copy number is produced.
