- Volume 88, Issue 9, 2007

Recombinant vaccinia viruses based on the attenuated NYVAC and MVA strains are promising vaccine candidates against a broad spectrum of diseases. Whilst these vectors are safe and immunogenic in animals and humans, little is known about their comparative behaviour in vivo. In this investigation, a head-to-head analysis was carried out of virus dissemination in mice inoculated by the mucosal or systemic route with replication-competent (WRluc) and attenuated recombinant (MVAluc and NYVACluc) viruses expressing the luciferase gene. Bioluminescence imaging showed that, in contrast to WRluc, the attenuated recombinants expressed the reporter gene transiently, with MVAluc expression limited to the first 24 h and NYVACluc giving a longer signal, up to 72 h post-infection, for most of the routes assayed. Moreover, luciferase levels in MVAluc-infected tissues peaked earlier than those in tissues infected by NYVACluc. These findings may be of immunological relevance when these vectors are used as recombinant vaccines.

Expression of chiA and v-cath RNA and enzyme activity in wild-type Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was compared with that of recombinant AcMNPV viruses reprogrammed for expression of the endogenous chiA. To establish a baseline for our recombinant AcMNPV studies, we compared, for the first time, the temporal expression profiles of both AcMNPV chiA transcription and translation simultaneously. The rate of intracellular chitinase accumulation during AcMNPV infection followed the same pattern observed for chiA transcription but was delayed by about 6 h. Replacement of 21 nucleotides containing the native late chiA and v-cath promoters with a selectable polh–EGFP cassette was sufficient to eliminate expression of both chiA and v-cath. Viruses were generated that express chiA from either the late p6.9 or very late polh promoters of AcMNPV, replacing the native chiA promoter. There was a marked difference in the temporal chiA transcription profiles from the native, p6.9 and polh promoters, resulting in respective specific activities of chitinase at 48 h p.i. of 62, 160 and 219 mU (mg lysate total protein)−1. Based on temporal analysis of v-cath transcription by Northern blot, AcMNPV v-cath was transcribed from 9 h p.i. in Sf21 cells. However, expression of v-cath RNA or enzyme from a reconstructed v-cath promoter in the chiA-reprogrammed viruses was not detected at 48 h of virus replication. Reprogramming for increased chitinase (and putatively cathepsin) expression with native baculovirus promoters might provide a means for designing environmentally benign biological insecticides.

The delayed-early DNA polymerase promoter of Chilo iridescent virus (CIV), officially known as Invertebrate iridescent virus, was fine mapped by constructing a series of increasing deletions and by introducing point mutations. The effects of these mutations were examined in a luciferase reporter gene system using Bombyx mori cells transfected with promoter constructs and infected with CIV. When the size of the upstream element was reduced from position −19 to −15, relative to the transcriptional start site, the luciferase activity was reduced to almost zero. Point mutations showed that each of the 5 nt (AAAAT) located between –19 and –15 were equally essential for promoter activity. Mutations at individual bases around the transcription initiation site showed that the promoter extended until position −2 upstream of the transcription start site. South-Western analysis showed that a protein of approximately 100 kDa interacted with the −19 nt promoter fragment in CIV-infected cells. This binding did not occur with a point mutant that lacked promoter activity. The AAAAT motif was also found in the DNA polymerase promoter region of other iridoviruses and in other putative CIV delayed-early genes.

A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LMV diagnostic and field epidemiology studies.

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

Dengue is caused by a taxonomic group of four viruses, dengue virus types 1–4 (DENV1–DENV4). A molecular understanding of the antibody-mediated protection against this disease is critical to design safe vaccines and therapeutics. Here, the energetic epitope of antibody mAb4E11, which neutralizes the four serotypes of DENV but no other flavivirus, and binds domain 3 (ED3) of their envelope glycoprotein, was characterized. Alanine-scanning mutagenesis of the ED3 domain from serotype DENV1 was performed and the affinities between the mutant domains and the Fab fragment of mAb4E11 were measured. The epitope residues (307–312, 387, 389 and 391) were at the edges of two distinct β-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency.

West Nile virus (WNV) is a mosquito-borne flavivirus that was first introduced into the USA in the New York City area in 1999. Since its introduction, WNV has steadily increased both its host and geographical ranges. Outbreaks of the closely related flavivirus, St. Louis encephalitis virus (SLEV), occur in the USA periodically, but levels of activity and host range are more restricted than those of WNV. Understanding the selective pressures that drive arbovirus adaptation and evolution in their disparate mosquito and avian hosts is crucial to predicting their ability to persist and re-emerge. Here, we evaluated the in vivo phenotypes of mosquito cell-adapted WNV and SLEV. Results indicated that in vitro adaptations did not translate to in vivo adaptations for either virus, yet SLEV displayed attenuated growth in both mosquitoes and chickens, while WNV generally did not. In vitro growth analyses also indicated that WNV adaptations could be generalized to cell cultures derived from other mosquito species, while SLEV could not. Analysis of genetic diversity for passaged SLEV revealed a highly homogeneous population that differed significantly from previous results of high levels of diversity in WNV. We hypothesize that this difference in genetic diversity is directly related to the viruses' success in new and changing environments in the laboratory and that differences in a viruses' ability to produce and maintain heterogeneous populations in nature may in some instances explain the variable levels of success seen among arboviruses.