Noboru Yanagida

Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus

Abstract Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Mareks disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody–positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gI were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Mareks disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

Recombinant fowlpox viruses inducing protective immunity against Newcastle disease and fowlpox viruses.

The haemagglutinin-neuraminidase gene of Newcastle disease virus was inserted into a non-essential region of the fowlpox virus genome and expressed under control of the vaccinia virus 7.5 kDa polypeptide gene promoter. Immunization with the recombinant fowlpox virus elicited protective immunity in chickens against both virulent Newcastle disease and fowlpox virus infection.

Nucleotide and predicted amino acid sequences of Marek's disease virus homologues of herpes simplex virus major tegument proteins

The DNA sequence of an 8.4 kbp BamHI-EcoRI fragment of Mareks disease virus (MDV) strain GA was determined. Three of the predicted polypeptides are homologous to UL47, UL48 and UL49 encoding the major tegument proteins of herpes simplex virus type 1 (HSV-1), and four are homologous to HSV-1 UL45, UL46, UL49.5 and UL50. These seven genes are found in the long unique region of the MDV genome and are collinear with homologues in HSV-1 and varicella-zoster virus (VZV). Northern blot analysis revealed different transcriptional patterns from those of HSV-1 and VZV. MDV homologues of UL49.5, UL49 and UL47 lack a poly(A) signal immediately downstream of their coding regions. Amino acid conservation between MDV and HSV-1, and between MDV and VZV is as high as that between HSV-1 and VZV. The MDV homologue of UL48 shows 60% similarity to its HSV-1 counterpart. Amino acid sequence comparison reveals that the MDV homologue of UL48 lacks an acidic carboxyl terminus. This homologue, like the VZV homologue of UL48, may be involved in the trans-activation of immediate early genes and may function as an important component of the structural proteins.

Antibody Response to Newcastle Disease Virus (NDV) of Recombinant Fowlpox Virus (FPV) Expressing a Hemagglutinin-Neuraminidase of NDV into Chickens in the Presence of Antibody to NDV or FPV

Antibody response of recombinant fowlpox virus (FPV) was studied in chickens inoculated with the virus in the presence or absence of antibodies against Newcastle disease virus (NDV) or FPV. In the case of NDV, high hemagglutination-inhibition titers to NDV were obtained when the antibody was present. No immune response to NDV was observed in the chickens previously vaccinated with FPV.

Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene.

A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.

Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions.

Insertion of the Escherichia coli lacZ gene into a ClaI restriction enzyme site of a 5.7 kb HindIII fragment of the fowlpox virus (FPV) genome resulted in the generation of stable recombinants. These recombinants produced plaques that were significantly smaller than those produced by parental FPV or by FPV recombinants containing the lacZ gene at other non-essential sites. Insertion of foreign DNA into the ClaI site disrupts a previously unidentified open reading frame (ORF) which potentially encodes a 74K polypeptide. The predicted amino acid sequence of this FPV ORF has 24% identity with the F12L ORF of vaccinia virus, the function of which is not currently known. Production of intracellular FPV was similar in cells infected with recombinant or parental viruses, but the number of infectious extracellular virions released into the medium by the recombinant was about 20% of that released by the parental virus. Likewise, the release of FPV particles, which were labelled in vivo with [3H]thymidine, was significantly lower in recombinant FPV-infected cells. These results suggest that the FPV homologue of the vaccinia virus F12L ORF is involved in the envelopment or release of infectious extracellular virions.

The Efficacy of Recombinant Fowlpox Vaccine Protection Against Marek's Disease: Its Dependence on Chicken Line and B Haplotype

Abstract Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Mareks disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%–97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 × 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%–100%) following either MDV challenge, but protection was significantly lower in 15 × 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 × 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.

A Recombinant Fowlpox Virus Expressing the Envelope Antigen of Subgroup A Avian Leukosis/Sarcoma Virus

A recombinant fowlpox virus (FPV) was constructed by inserting cloned sequences from Schmidt-Ruppin subgroup A avian sarcoma virus coding for the viral envelope (env) antigen into a nonessential region of FPV DNA downstream from a synthetic promoter. Sera from chickens hyperimmunized with the recombinant FPV neutralized the infectivity of the homologous subgroup A virus (RCASBP/AP) but only weakly neutralized the infectivity of Rous sarcoma virus, another subgroup A avian leukosis virus. Similarly, vaccination of 1-day-old chicks with this recombinant FPV protected against infection with RCASBP/AP virus but not against infection with another subgroup A Rous-associated virus (RAV-1). These results show that such a recombinant FPV can be used to protect chickens against avian leukosis virus and confirm previous observations that a type-specific antigenic variability existed within the subgroup A avian leukosis/sarcoma virus group.

Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek's disease virus.

The Mareks disease virus (MDV) glycoprotein B (gB) precursor, gp100, is proteolytically cleaved into two disulfide-linked subunits, gp60 and gp49. In the gB homologs of most other herpesviruses, a tetrapeptide, Arg-Xaa-Arg-Arg, is immediately upstream from the predicted cleavage site. We have investigated the specificity of the proteolytic cleavage in gp100 by introducing mutations within its predicted cleavage site (Arg-Leu-Arg-Arg) and expressed these mutants in recombinant fowlpox virus (FPV). The results show that all three Arg residues at the predicted cleavage site play an important role in the specific proteolytic cleavage of gp100. Furthermore, we demonstrated that the cleavage of gp100 is not necessary for transport of gB to the cell surface.