- Volume 87, Issue 10, 2006

The subviral dodecahedral particle of adenovirus 3, which assembles spontaneously in insect cells expressing the viral penton base protein, shows promise as a vector for drug delivery. Its ability to gain cell entry has been demonstrated and recent structural analysis has outlined details of the interfaces between penton bases and the importance of proteolysis of the penton base N terminus for assembly, providing a basis for understanding particle assembly and stability. Here, work in manipulating the assembly status of the dodecahedron by changing buffer conditions and subsequent success in passively encapsidating a marker molecule is described. This represents an important stage towards development of the dodecahedral particle for use as a delivery vehicle capable of targeting therapeutic molecules to specific cell types.

Singapore grouper iridovirus (SGIV), an iridovirus in the genus Ranavirus, is a major pathogen that results in significant economic losses in grouper aquaculture. To investigate further its infective mechanisms, for the first time, a viral DNA microarray was generated for the SGIV genome to measure the expression of its predicted open reading frames simultaneously in vitro. By using the viral DNA microarray, the temporal gene expression of SGIV was characterized and the DNA microarray data were consistent with the results of real-time RT-PCR studies. Furthermore, different-stage viral genes (i.e. immediate-early, early and late genes) of SGIV were uncovered by combining drug treatments and DNA microarray studies. These results should offer important insights into the replication and pathogenesis of iridoviruses.

Safety-tested vaccinia virus (VACV) MVA serves as a candidate third-generation vaccine against smallpox. Here, MVA immunization of mice shortly before or after lethal respiratory challenge with VACV Western Reserve was investigated. Whilst post-exposure treatment failed to protect animals, immunizations on day 2 prior to challenge were fully protective. On the day of challenge, MVA inoculation may prevent death, but not onset of severe respiratory disease. After intranasal MVA application, massive influx of leukocytes (such as neutrophils, macrophages, natural killer cells and T cells) was found in the lungs of the animals, indicating the contribution of innate responses to protection. Correspondingly, in RAG-1−/− mice, MVA inoculation delayed onset of disease significantly, but did not prevent fatal infection. Thus, short-term protection required a tight interplay of both innate and adaptive antiviral immunity. These data suggest that, in addition to conventional vaccination, MVA may serve for potent emergency prophylaxis against orthopoxvirus infection.

There are marked differences in the clinical expression of diseases associated with porcine circovirus type 2 (PCV2) in the field. The objective of this study was to compare the sequences and pathogenicity of PCV2 isolates from field cases with and without PCV2-associated lesions. Forty-two specific-pathogen-free (SPF) pigs were assigned randomly to three groups of 14 pigs each. At 7 weeks of age, group 1 pigs were mock-inoculated with saline, group 2 pigs were inoculated with PCV2-4838 (isolated from a pig with no evidence of PCV2-associated lymphoid lesions) and group 3 pigs were inoculated with PCV2-40895 (isolated from a pig with PCV2-associated lymphoid lesions and disease). The PCV2-4838 and PCV2-40895 isolates shared approximately 98.9 % nucleotide sequence identity across the entire genome. A total of nine amino acid changes in ORF2 and two amino acid changes in ORF1 were identified between the two isolates. PCV2-4838-inoculated pigs had significantly more genomic copy numbers of PCV2 in their sera at 7 days post-inoculation (p.i.) (P<0.0001) and significantly fewer genomic copy numbers at 14, 21 and 28 days p.i. (P<0.05) compared with pigs inoculated with the PCV2-40895 isolate. Microscopic lesions in lymphoid tissues were significantly less severe (P<0.05) and the amount of PCV2 antigen associated with these lesions was significantly lower (P<0.05) in pigs inoculated with PCV2-4838. The results of this study suggest that PCV2 isolates from the USA differ in virulence in an SPF pig model.

The anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) was investigated in vitro in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells and in RSV-induced tumours of specific-pathogen-free (SPF) chicks in vivo. The apoptin gene was cloned in the pVAX expression vector and in vitro expression of the recombinant vector pVAX-CAV-VP3 was confirmed. Two groups of SPF chicks, each containing ten chicks, were used. Chicks in groups I and II were inoculated with RSV at 1 day old. Group I served as the control, receiving pVAX vector without insert, and group II received recombinant vector pVAX-CAV-VP3 containing the apoptin gene, on day 10. An in vitro study confirmed that apoptin induced apoptosis in RSV-transformed CEF cells, which was demonstrated by observation of the characteristic changes of apoptosis using the indirect immunofluorescence technique and acridine orange/ethidium bromide staining. In vivo study also indicated that apoptin induced apoptosis and caused tumour regression by an intratumoral-delivery method. Apoptotic changes, such as nuclear condensation, fragmentation of the chromatin and formation of apoptic bodies in the tumour cells, were demonstrated by histopathology and acridine orange/ethidium bromide staining. No apoptotic changes were seen in the tumours of the control group. The results of the present study showed that apoptin had an anti-neoplastic effect in vivo and in vitro in RSV-induced tumours. The anti-neoplastic effect is due to apoptin-induced apoptosis. Further improvements in the dose, delivery method and delivery frequency of the apoptin-expressing recombinant vector could help to develop apoptin as an anti-neoplastic drug.

Recently, three distinct genotypes (1, 2 and 3) of human parvovirus B19 (B19) have been identified. However, the characteristics and distribution of B19 genotypes in Vietnam have not been investigated. Phylogenetic analysis using 49 subgenomic NS1/VP1u regions and two coding NS1–VP1/VP2 regions has been applied to investigate the prevalence of B19 genotypes in Vietnamese patients co-infected with Hepatitis B virus. Genetic analysis of the subgenomic NS1/VP1u region of B19 revealed that two genotypes of B19 were identified in these populations, with predominance of genotype 1 (47/49, 96 %) followed by genotype 2 (2/49, 4 %), but not genotype 3. Further, phylogenetic analysis of subgenomic B19 genomes revealed two major subgroups within genotype 1 (B19-1A and B19-1B) with an estimated nucleotide difference of >5 % between each subgroup, forming different branches. The mean percentage of amino acid variation between subgroup B19-1A and B19-1B was >2 % of the NS1, VP1 and VP2 proteins. Our results indicated that two of the three known genotypes of B19 were present in Vietnamese patients, with genotype 1 predominating, and that this genotype can be classified into at least two subgroups, B19-1A and B19-1B.

Infection with BK virus (BKV) induces both humoral and cellular immunity, but the viral antigens of T-antigen (T-ag) stimulating T-cell responses are largely unknown. To identify BKV-specific T cells in healthy individuals, peripheral blood lymphocytes were cultured with autologous dendritic cells (DCs) loaded with BKV lysate and T cells were screened for intracellular gamma interferon production after stimulation with an overlapping 15mer peptide library of the BKV T-ag. Among many immunogenic peptides identified, four T-ag peptides were identified as candidate major histocompatibility complex class I and II T-cell epitopes, restricted to human leukocyte antigen (HLA)-B*0702, -B*08, -DRB1*0301 and -DRB1*0901. Further, a candidate 9mer peptide, LPLMRKAYL, was confirmed to be restricted to HLA-B*0702 and -B*08. Because the polyomaviruses BKV, JC virus (JCV) and Simian virus 40 (SV40) share extensive sequence similarity in the immunogenic proteins T-ag and VP1, it was hypothesized that, in humans, these proteins contain conserved cytotoxic T-lymphocyte (CTL) target epitopes. Four HLA-restricted conserved epitopes of BKV, JCV and SV40 were identified: HLA-B*07, -B*08 and -DRB1*0901 for T-ag and -A*0201 for VP1. T cells cultured in vitro that were specific for one viral antigen recognized other conserved epitopes. CTLs generated from BKV T-ag and VP1 peptide were cytotoxic to DC targets pulsed with either BKV or JCV. Therefore, infection by one of the two viruses (BKV and JCV) could establish cross-immunity against the other. Although cross-cytotoxicity experiments were not performed with SV40, cross-recognition data from conserved antigen epitopes of polyomaviruses suggest strongly that cross-immunity might also exist among the three viruses.

Numerous studies have documented molecular variability in plant virus populations, but few have assessed the relative contribution of natural selection and genetic drift in generating the observed pattern of diversity. To this end, gene function, environment and phylogenetic history were examined to observe the effect on genetic diversity and population structure of the PAV and PAS species of Barley yellow dwarf virus (family Luteoviridae). Three functional classes of gene were analysed: transcription-related (RdRp), structural (CP) and movement-related (MP). The results indicate that there were no inherent differences, in terms of total diversity or diversity at synonymous or non-synonymous nucleotide sites, between functional classes of genes or populations. Rather, selective constraints on a gene may be more or less relaxed depending on its function and the phylogenetic history of the population sampled. The CP of the PAS species, but not the PAV species, was differentiated genetically between regions. This is probably due to genetic drift, as there was no evidence that any gene deviated from a neutral model of evolution or is under positive selection. In general, the MP was under considerably less functional constraint than structural or replication-related proteins and four positively selected codon sites were identified. Mutations at these sites differentiate species and geographical subpopulations, so presumably they have aided the virus in adaptation to its host environment and contributed to intra- and interspecies diversification.

A full-length cDNA of the genome of Beet black scorch virus (BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permit in vitro transcription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G2209 within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G2526 within the nested p7b/p5′ ORF. Initiation-codon shifting or premature termination of translation of the three ORFs (P7a, P7b and P5′) encoded by sgRNA1 indicated that each of the genes was required for localized movement, accumulation of viral RNAs and formation of local lesions on the leaves of Chenopodium amaranticolor. Microscopic observations of the distribution of green fluorescent protein fused to the N-terminal portion of the capsid protein provided additional evidence that the P7a, P7b and P5′ proteins are each required for cell-to-cell movement. In contrast, elimination of sgRNA2 showed that the BBSV coat protein was not required for viral RNA accumulation or the appearance of local lesions on C. amaranticolor. In addition, disruption of the small P5 ORF previously predicted by computer analysis to originate at the C terminus of the P82 ORF had no effect on disease phenotype, suggesting that this ORF may represent a cryptic, non-essential gene. These results show that BBSV has a novel cell-to-cell movement protein organization that differs in size and sequence from that of other viruses.

The TGBp1 protein, encoded in the genomes of a number of plant virus genera as the first gene of the ‘triple gene block’, possesses an NTPase/helicase domain characterized by seven conserved sequence motifs. It has been shown that the TGBp1 NTPase/helicase domain exhibits NTPase, RNA helicase and RNA-binding activities. In this paper, we have analysed a series of deletion and point mutants in the TGBp1 proteins encoded by Potato virus X (PVX, genus Potexvirus) and Poa semilatent virus (PSLV, genus Hordeivirus) to map functional regions responsible for their biochemical activities in vitro. It was found that, in both PVX and PSLV, the N-terminal part of the TGBp1 NTPase/helicase domain comprising conserved motifs I, Ia and II was sufficient for ATP hydrolysis, RNA binding and homologous protein–protein interactions. Point mutations in a single conserved basic amino acid residue upstream of motif I had little effect on the activities of C-terminally truncated mutants of both TGBp1 proteins. However, when introduced into the full-length NTPase/helicase domains, these mutations caused a substantial decrease in the ATPase activity of the protein, suggesting that the conserved basic amino acid residue upstream of motif I was required to maintain a reaction-competent conformation of the TGBp1 ATPase active site.

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, has a segmented, negative-stranded RNA genome. We examined the population structure and genetic variation of CPsV in three coding regions located in RNAs 1, 2 and 3, analysing 22 isolates from Argentina, California, Florida, Italy and Spain. Most isolates contained a predominant sequence and some minor variants. Estimations of the genetic diversity and phylogenetic clustering of isolates disclosed two populations, one comprising isolates from Spain, Italy, Florida and California and the other including the Argentinean isolates. Isolate CPV-4 (from Texas) included for comparison was distant from both groups, suggesting that it belongs to a third group. The low ratio between non-synonymous and synonymous nucleotide substitutions indicated strong selection for amino acid sequence conservation, particularly in the coat protein gene. Incongruent phylogenetic relationships in different genomic regions suggested that exchange of genomic segments may have contributed to CPsV evolution.

The potexvirus Potato virus X (PVX) can be modified genetically to generate chimeric virus particles (CVPs) carrying heterologous peptides fused to coat protein (CP) subunits. A spontaneous PVX mutant expressing a truncated, but functional, form of the CP has been isolated. With the aim of exploiting this virus to display peptides useful for vaccine formulations, two novel viral expression vectors based on pPVX201 (bearing the wild-type PVX genome) were constructed encoding the truncated CP. Both vectors were able to produce infectious virus particles in planta and were used to insert a panel of sequences encoding peptides of biopharmaceutical interest as N-terminal fusions to the truncated cp gene. The analysis of infection progression induced by the different constructs enabled identification of two important structural features of the fused peptide, namely tryptophan content and isoelectric point, critically affecting the formation of PVX CVPs and virus movement through the plant. These results are discussed in view of the rising interest in engineered plant viruses for development of peptide-based epitope vaccines.

Prions are postulated to be the infectious agents of a family of transmissible, fatal, neurodegenerative disorders affecting both humans and animals. The possibility of prion transmission constitutes a public-health risk that confronts regulatory authorities everywhere. The main problem in handling prions is the fact that they are extremely resistant to standard decontamination methods. Thus, the use of harsh and expensive practices to destroy prions is inevitable. The development of applicable and efficient prion-inactivation practices is still highly important for the prevention of accidental transmission. In the search for effective and environmentally friendly methods to eliminate organic compounds and bacteria, much attention has been focused on the so-called advanced oxidation processes. These are based on the formation of hydroxyl radicals, which are known to possess a high reductive potential. This study tested the potential of titanium dioxide, an inexpensive and completely inert reagent, to inactivate prions in a heterogeneous photocatalytic process. Initial in vitro experiments were followed by a bioassay with the scrapie strain 263K in Syrian hamsters. The results obtained from this study indicate that titanium dioxide photocatalytic treatment of scrapie-infected brain homogenates reduces infectivity titres significantly.

To gain insight into the role of cell surface glycosaminoglycans (GAG) in dengue virus (DEN) cell tropism and virulence, DEN-2 mouse brain-adapted vaccine candidate, neurovirulent prototype strain (NGC) and low-passage strain, PUO-218, were passaged in BHK-21 and SW13 cells to isolate variants with high affinity for GAG. Sequence comparisons of parent and passage variants revealed five GAG-binding determinants, which all cluster in a surface-exposed region in domain II of the three-dimensional structure of the DEN envelope protein. Using an infectious cDNA clone of NGC and an NGC/PUO-218 prM–E chimeric clone, it was demonstrated that the GAG-binding determinants augment the specific infectivity for BHK-21 and/or SW13 cells by 10- to 170-fold and in some cases marginally reduce that for Vero cells. This altered cell tropism was due to a greater dependence of the variants on cell surface GAG for attachment/entry, given their increased susceptibility to heparin inhibition. The effect of the GAG-binding determinants on virulence was examined in mice deficient in alpha/beta/gamma interferon responses. High GAG affinity strongly correlated with low neuroinvasiveness due to rapid virus clearance from the blood. It was speculated that this mechanism accounts for the attenuation in primates of some DEN vaccine candidates. Interestingly, the GAG-binding variants did not display marked attenuation of neurovirulence and the opposing effect of enhanced neurovirulence was associated with one determinant (Lys126) already present in mouse brain-adapted NGC. This discrepancy of attenuated neuroinvasiveness and augmented neurovirulence may be reconciled by the existence of different mechanisms of virus dissemination in the brain and in extraneural tissues.

Reassortment among the RNA segments of Influenza A virus caused the two most recent human influenza pandemics; recently, reassortment has generated viral genotypes associated with outbreaks of avian H5N1 influenza in Asia and Europe. A statistical analysis has been developed for the systematic identification and characterization of reassortant viruses. The analysis was applied to the genes of the replication complex of 152 avian influenza A viruses isolated between 1966 and 2004 from predominantly terrestrial and domestic aquatic avian species. The results indicated that reassortment among these genes was pervasive throughout this period and throughout both the Eurasian and North American lineages of the virus. Evidence is presented that the circulating genotypes of the replication complex are being replaced continually by novel genotypes created by reassortment. No constraints for coordinated reassortment among genes of the replication complex were evident; rather, reassortment almost always proceeded one segment at a time. A maximum-likelihood estimate of the rate of reassortment was derived. For significantly diverged Asian avian influenza A viruses from the period 1991–2004, it was estimated that the median duration between creation of a new genotype and its next segment reassortment was 3 years. Reassortments that introduced previously unobserved influenza genetic material were detected. These findings point to substantial potential for rapid generation of novel avian influenza A viruses, emphasizing the importance of intensive surveillance of these host species in preparation for a possible pandemic.