- Volume 83, Issue 10, 2002

A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek’s disease virus (MDV) was tested for its potential to protect against Marek’s disease (MD). Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery. Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection. A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird. Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA. In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection. In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus. The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection.

Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition of oriP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP–EBNA-1) is examined by confocal microscopy combined with a ‘premature chromosome condensation’ (PCC) procedure. Analyses show that GFP–EBNA-1 expressed in living cells that lack oriP plasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP–EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.

The primary structure of a novel envelope protein from shrimp white spot syndrome virus (WSSV) was characterized using a combination of SDS–PAGE and mass spectrometry. The resulting amino acid sequence matched an open reading frame (ORF), ORF1050, of the WSSV genome ORF database. ORF1050 contained 843 nt, encoding 281 aa, and was termed the vp281 gene. Computer-assisted analysis showed that both the vp281 gene and its product shared no significant homology with other known viruses. However, they shared striking identity/similarity with another WSSV structural protein, VP292, at both the nucleotide and amino acid sequence level, suggesting that vp281 and vp292 might have evolved by gene duplication from a common ancestral gene. WSSV VP281 cDNA was cloned into a pET32a(+) expression vector containing a T7 RNA polymerase promoter to produce (His)6-tagged fusion proteins in Escherichia coli strain BL21. Specific mouse antibodies were raised using the purified fusion protein (His)6-VP281. Western blot analysis showed that the mouse anti-(His)6-VP281 antibodies bound specifically to VP281 of WSSV, without cross-reactivity with VP292. The transmission electron microscope immunogold-labelling method was used to localize VP281 in the WSSV virion as an envelope protein. The cell attachment ‘Arg–Gly–Asp’ motif in VP281 indicated that this protein might play an important role in mediating WSSV infectivity.

Campoletis sonorensis ichnovirus (CsIV) is a symbiotic virus associated with the endoparasitic wasp C. sonorensis. The virus is injected into the wasp’s host, Heliothis virescens, during oviposition. One CsIV gene has been identified as a repeat element (rep) gene and encodes a ubiquitous imperfectly conserved 540 bp sequence. We report the sequencing and mapping of a rep-containing segment, segment I, that hybridizes to a known rep sequence from segment O1. Analysis of this 8·6 kbp segment identified three ORFs having high similarity to the 540 bp rep sequence. All three rep sequence ORFs were expressed in parasitized H. virescens as well as in C. sonorensis tissues. Two of these rep genes, I 0.9 and I 1.1, have single copies of the 540 bp repeat sequence, while the third rep gene, I 1.2, has two imperfect copies, which are more similar to each other than to sequences on the segment I single-motif genes. Like the CsIV BHv 0.9 rep gene, the segment I rep genes lack introns and a signal peptide, suggesting that they are not secreted. Based on their similarity in nucleotide sequence, predicted amino acid sequence and gene structure, the three segment I repeat-containing genes, I 0.9, I 1.1 and I 1.2, are new members of the rep gene family.

Approximately 60% of the genome of an adenovirus isolated from a corn snake (Elaphe guttata) was cloned and sequenced. The results of homology searches showed that the genes of the corn snake adenovirus (SnAdV-1) were closest to their counterparts in members of the recently proposed new genus Atadenovirus. In phylogenetic analyses of the complete hexon and protease genes, SnAdV-1 indeed clustered together with the atadenoviruses. The characteristic features in the genome organization of SnAdV-1 included the presence of a gene homologous to that for protein p32K, the lack of structural proteins V and IX and the absence of homologues of the E1A and E3 regions. These characteristics are in accordance with the genus-defining markers of atadenoviruses. Comparison of the cleavage sites of the viral protease in core protein pVII also confirmed SnAdV-1 as a candidate member of the genus Atadenovirus. Thus, the hypothesis on the possible reptilian origin of atadenoviruses (Harrach, Acta Veterinaria Hungarica 48, 484–490, 2000) seems to be supported. However, the base composition of DNA sequence (>18 kb) determined from the SnAdV-1 genome showed an equilibrated GC content of 51%, which is unusual for an atadenovirus.

The potyvirus Papaya ringspot virus (PRSV) is found throughout the tropics and subtropics. Its P biotype is a devastating pathogen of papaya crops and its W biotype of cucurbits. PRSV-P is thought to arise by mutation from PRSV-W. However, the relative impact of mutation and movement on the structure of PRSV populations is not well characterized. To investigate this, we have determined the coat protein sequences of isolates of both biotypes of PRSV from Vietnam (50), Thailand (13), India (1) and the Philippines (1), and analysed them together with 28 PRSV sequences already published, so that we can better understand the molecular epidemiology and evolution of PRSV. In Thailand, variation was greater among PRSV-W isolates (mean nucleotide divergence 7·6%) than PRSV-P isolates (mean 2·6%), but in Vietnamese populations the P and W biotypes were more but similarly diverse. Phylogenetic analyses of PRSV also involving its closest known relative, Moroccan watermelon mosaic virus, indicate that PRSV may have originated in Asia, particularly in the Indian subcontinent, as PRSV populations there are most diverse and hence have probably been present longest. Our analyses show that mutation, together with local and long-distance movement, contributes to population variation, and also confirms an earlier conclusion that populations of the PRSV-P biotype have evolved on several occasions from PRSV-W populations.

The population structure and genetic diversity of Citrus leaf blotch virus (CLBV) were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of two genomic regions located within the replicase (R) and the coat protein (C) genes. Analysis of 30 cDNA clones of each genomic region from two CLBV isolates showed that both isolates contained a predominant haplotype and others closely related. Analysis of 37 CLBV Spanish field isolates showed low genetic diversity (0·0041 and 0·0018 for genomic regions R and C, respectively). Comparison of 14 CLBV isolates from Spain, Japan, USA, France and Australia showed genetic diversities of 0·0318 (R) and 0·0209 (C), respectively. No correlation was found between genetic distance and geographical origin or host species of the isolates. The ratio between nonsynonymous and synonymous substitutions was the lowest found in a plant virus, indicating a strong negative selective pressure in both genomic regions.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle χ2,1 for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

Up to 15% of free-ranging mule deer in northeastern Colorado and southeastern Wyoming, USA, are afflicted with a prion disease, or transmissible spongiform encephalopathy (TSE), known as chronic wasting disease (CWD). CWD is similar to a subset of TSEs including scrapie and variant Creutzfeldt–Jakob disease in which the abnormal prion protein isoform, PrPCWD, accumulates in lymphoid tissue. Experimental scrapie studies have indicated that this early lymphoid phase is an important constituent of prion replication interposed between mucosal entry and central nervous system accumulation. To identify the lymphoid target cells associated with PrPCWD, we used triple-label immunofluorescence and high-resolution confocal microscopy on tonsils from naturally infected deer in advanced disease. We detected PrPCWD primarily extracellularly in association with follicular dendritic and B cell membranes as determined by frequent co-localization with antibodies against membrane bound immunoglobulin and CD21. There was minimal co-localization with cytoplasmic labels for follicular dendritic cells (FDC). This finding could indicate FDC capture of PrPCWD, potentially in association with immunoglobulin or complement, or PrPC conversion on FDC. In addition, scattered tingible body macrophages in the germinal centre contained coarse intracytoplasmic aggregates of PrPCWD, reflecting either phagocytosis of PrPCWD on FDC processes, apoptotic FDC or B cells, or actual PrPCWD replication within tingible body macrophages. To compare lymphoid cell targets in early and advanced disease, we also examined: (i) PrPCWD distribution in lymphoid cells of fawns within 3 months of oral CWD exposure and (ii) tonsil biopsies from preclinical deer with naturally acquired CWD. These studies revealed that the early lymphoid cellular distribution of PrPCWD was similar to that in advanced disease, i.e. in a pattern suggesting FDC association. We conclude that in deer, PrPCWD accumulates primarily extracellularly and associated with FDCs and possibly B cells – a finding which raises questions as to the cells responsible for pathological prion production.

The usefulness of tonsillar biopsy on live deer for preclinical diagnosis of the transmissible spongiform encephalopathy chronic wasting disease (CWD) was evaluated. Disease was tracked in a CWD-endemic herd using serial tonsillar biopsies collected at 6 to 9 month intervals from 34 captive mule deer (Odocoileus hemionus) and five white-tailed deer (O. virginianus). Tonsillar biopsies were examined for accumulation of PrPCWD, the protein marker for infection, using immunohistochemical (IHC) staining. 26/34 (76%) mule deer and 4/5 (80%) white-tailed deer had PrPCWD accumulation in tonsillar biopsies; CWD was subsequently confirmed by post-mortem examination in all 30 of these tonsillar-positive deer. Six mule deer with IHC-negative tonsillar biopsies had positive brain and tonsillar IHC staining upon death 12 to 40 months following the last biopsy. PrPCWD accumulation in tonsillar biopsy was observed 2 to 20 months before CWD-related death and up to 14 months before onset of clinical signs of CWD. Tonsillar biopsies from 3-month-old mule deer (n=6) were IHC negative, but PrPCWD accumulation was detected in tonsillar biopsies from 7/10 mule deer by 19 months of age. Tonsillar biopsy evaluated with IHC staining is a useful technique for the preclinical diagnosis of CWD in live mule deer and white-tailed deer when intensive management approaches are possible.