Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

The detection by serological methods of viruses infecting the rose

Homogenates of herbaceous test plants infected with arabis mosaic virus (AMV), prunus necrotic ringspot virus (PNRSV), or strawberry latent ringspot virus (SLRV), and purified virus preparations were used to assess the sensitivities of four serological methods (the enzyme-linked immunosorbent assay - ELISA, immunodiffusion in gels, the latex flocculation assay, and serologically specific electron microscopy -SSEM) for the detection of these viruses. The latex test was up to 250 times more sensitive than gel immunodiffusion, but SSEM and ELISA were respectively up to 1000 and 200 times more sensitive than the latex test. Gel immunodiffusion and latex tests failed to detect any of the viruses in infected roses. Although ELISA reliably detected PNRSV and SLRV when leaves from infected roses were homogenised in a leaf: buffer ratio of 1 g:10 ml, AMV was occasionally undetected. However, when a modified ELISA technique, which reduced non-specific reactions, was used some PNRSV-infected roses were also not detected. Detection by SSEM was c. twice as sensitive as ELISA for all three viruses in rose extracts. The relative advantages of ELISA and SSEM for the detection of plant viruses are discussed.     

Genome wide survey of microsatellites in ssDNA viruses infecting vertebrates

Microsatellites or Simple Sequence Repeats (SSRs) are tandem iterations of one to six base pairs, non-randomly distributed throughout prokaryotic and eukaryotic genomes. Limited knowledge is available about distribution of microsatellites in single stranded DNA (ssDNA) viruses, particularly vertebrate infecting viruses. We studied microsatellite distribution in 118 ssDNA virus genomes belonging to three families of vertebrate infecting viruses namely Circoviridae, Parvoviridae, and Anelloviridae, and found that microsatellites constitute an important component of these virus genomes. Mononucleotide repeats were predominant followed by dinucleotide and trinucleotide repeats. A strong positive relationship existed between number of mononucleotide repeats and genome size among all the three virus families. A similar relationship existed for the occurrence of DTTPH (di-, tri-, tetra-, penta- and hexa-nucleotide) repeats in the families Anelloviridae and Parvoviridae only. Relative abundance and relative density of mononucleotide repeats showed a strong positive relationship with genome size in Circoviridae and Parvoviridae. However, in the case of DTTPH repeats, these features showed a strong relationship with genome size in Circoviridae only. On the other hand, relative microsatellite abundance and relative density of mononucleotide repeats were negatively correlated with GC content (%) in Parvoviridae genomes. On the basis of available annotations, our analysis revealed maximum occurrence of mononucleotide as well as DTTPH repeats in the coding regions of these virus genomes. Interestingly, after normalizing the length of the coding and non-coding regions of each virus genome, we found relative density of microsatellites much higher in the non-coding regions. We understand that the present study will help in the better characterization of the stability, genome organization and evolution of these virus classes and may provide useful leads to decipher the etiopathogenesis of these viruses.     

New members of a group of DNA viruses infecting brown algae

Since 1990 virus infections have been described in six brown algal species of the genera Ectocarpus, Feldmannia, Hincksia and Myriotrichia. These pathogens can be experimentally transmitted to healthy isolates of their hosts. A synopsis including new molecular and biochemical data shows that these viruses share common characteristics: genomes of double-stranded DNA, infection mode, morphology, extended temperence, and narrow host-specificity. These properties distinguish the brown algal viruses from all other known plant viruses.     

Live-cell microarray surface coatings supporting reverse transduction by adeno-associated viruses

High-throughput live-cell microarray technologies that facilitate combinatorial screening of genes and RNA interference (RNAi) would be invaluable in the identification of key gene expression profiles involved in complex cellular behaviors. Each spot on such a microarray can comprise a unique combination of genes or RNAi packaged into gene delivery vectors. Live target cells seeded on top of the microarrays would express the combination of genetic factors, potentially leading to phenotypic changes within cells. Here, we investigate the feasibility of using adeno-associated virus (AAV) as a gene delivery agent for such live-cell genetic microarrays. A robotic spotter was used to deposit AAV onto gamma-amino propyl silane, amine silane, or nitrocellulose-coated glass slides. Virus deposition and reverse transduction of target cells were found to be surface coating-dependent with nitrocellulose coating yielding the best AAV deposition, while also producing discrete islands of highly transduced cells. Our results demonstrate the feasibility of using nitrocellulose-coated surfaces for the development of AAV-based genetic microarrays.     

Stable gene expression by self-complementary adeno-associated viruses in human MSCs

Genetically modified mesenchymal stem cells (MSCs) are potentially valuable tools for the novel treatment of human illnesses. Here, we investigated whether gene transfers by self-complementary adeno-associated viruses (scAAV) lead to promising genetic modification in human bone marrow and umbilical cord blood MSCs. Of the various scAAVs, scAAV2, and scAAV5 effectively and safely expressed transgenes in both hMSCs. Transduction efficiency with scAAV2 at 1000 multiplicity of infection was 66.3+/-9.4% and 67.6+/-6.7% in bone marrow and umbilical cord blood MSCs, respectively. A co-infection study showed that the distinct scAAV2 and scAAV5 can effectively express different transgenes in the same hMSC. hMSCs transduced by scAAVs showed long-term gene expression for three months in rat brains. Genetic modification by scAAVs did not affect osteogenic differentiation of hMSCs. Therefore, the present study strongly supports the promising potential of scAAVs as a technical platform for safe, long-term transgene expression in hMSCs.     

Epidemiology of three viruses infecting the rose in the United Kingdom

Studies on the epidemiology of arabis mosaic (AMV), prunus necrotic ringspot (PNRSV) and strawberry latent ringspot (SLRV) viruses were made in relation to commercial production of standard and bush roses. AMV or SLRV apparently induced either symptomless infection in rose cultivars and Rosa spp., or leaf symptoms ranging from small chlorotic flecks to severe chlorotic mosaic and, occasionally, plant death. Infection of R. canina ‘inermis’ or R. corymbifera by an isolate of SLRV from R. corymbifera also severely depressed flowering and hip formation. In addition, whereas this isolate could be graft-transmitted to all Rosa spp. tested, isolates from R. rugosa and R. multiflora failed to be graft-transmitted to R. canina ‘inermis’ or R. corymbifera. No difference was detected in graft-transmission tests of Rosa spp. with several isolates of AMV or PNRSV. In plantings of up to 7 yr none of the viruses was transmitted through pollen to healthy roses grown in nematode-free soil, and only SLRV was readily seed-transmitted, particularly in R. rugosa. Nevertheless, in soil containing viruliferous nematodes, AMV and/or SLRV were transmitted to c. 80% of healthy plants. AMV and particularly SLRV were each damaging to field-grown maiden rose bushes cv. Fragrant Cloud. SLRV delayed the onset of flowering, and reduced the number and size of blooms. Diseased bushes were less vigorous, and half or none of the AMV- or SLRV- infected bushes respectively, conformed to the British Standards Institution specifications for maiden bush roses. These results are discussed in relation to the commercial production of field-grown roses in the UK.     

Diagnosis of viruses infecting essential-oil plant crops by using immunoenzyme analysis

The use of the EIA point technique permitted the detection of viruses affecting essential-oil plants. The method is simple, highly specific, sensitive and can be used for checking seedlings prior to planting.     

Isolation and characterization of phages infecting Bacillus cereus

Aim: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. Methods and Results: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single‐step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform‐resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log₁₀ CFU ml^?1 within 24 h at room temperature, when applied at a high concentration. Conclusions: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a ‘cocktail’ of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. Significance and Impact of the Study: This is the first report of biocontrol by phages of B. cereus in food.     

Isolation and characterization of bacteriophages infecting Salmonella spp

Bacteriophages infecting Salmonella spp. were isolated from sewage using soft agar overlays containing three Salmonella serovars and assessed with regard to their potential to control food-borne salmonellae. Two distinct phages, as defined by plaque morphology, structure and host range, were obtained from a single sample of screened sewage. Phage FGCSSa1 had the broadest host range infecting six of eight Salmonella isolates and neither of two Escherichia coli isolates. Under optimal growth conditions for S. Enteritidis PT160, phage infection resulted in a burst size of 139 PFU but was apparently inactive at a temperature typical of stored foods (5 degrees C), even at multiplicity of infection values in excess of 10 000. While neither isolate had characteristics that would make them candidates for biocontrol of Salmonella spp. in foods, phage FGCSSa1 behaved unusually when grown on two Salmonella serotypes at 37 degrees C in that the addition of phages appeared to retard growth of the host, presumably by the lysis of a fraction of the host cell population.     

Molecular diagnosis of medical viruses

The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories.     

Molecular ecology of microalgal viruses

A great amount of virus particles exist in natural waters. Each virion is considered to have its own ecological role, affecting the maintenance and fluctuation of aquatic ecosystems. We have been studying viruses infectious to micro-plankton, especially those infecting phytoplankton. Red tides are caused by drastic increase in abundance of plankton. We succeeded in elucidating that viral infection is one of the most important factors determining the dynamics and termination of algal blooms by means of field survey and molecular experiments. In addition, we demonstrated that the interrelationship between viruses and their hosts are highly complicated, and might be determined by the molecular-structural difference of viral capsids among distinct virus ecotypes. Furthermore, in the process of our investigation on various aquatic algal viruses, their importance as genetic sources has also been suggested. In order to deeply understand the mechanism of aquatic ecosystem, more intensive studies as for aquatic viruses are urgently required.     

Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses

Background

La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.

Results

To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.

Conclusions

SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.     

Cloning and characterization of a bovine adeno-associated virus

To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.     

Molecular phylogeny of geminivirus infecting wild plants in Japan

Few studies have been made on the molecular divergence of plant viruses. To remedy this deficiency, we examined the molecular divergence of the tobacco leaf curl geminivirus (TLCV). TLCV infects not only tobacco but alsoEupatorium andLonicera in the field and causes yellow vein disease. A total of 29 nucleotide sequences of the replication protein gene (ORF C1) of geminiviruses infecting wild plants ofE. makinoi, E. glehni andL. japonica collected from ten localities was determined. Highly divergent sequences were obtained not only among host plant populations but also within a host population. Phylogenetic analyses showed that the TLCVs infectingEupatorium andLonicera were clustered into three different clades, and were either paraphyletic or polyphyletic. This result is the first evidence demonstrating that wild populations of single plant species possess genetically diversified virus strains. Comparison with recently reported genetic variations of tobacco mild green mosaic tobamovirus (TMGMV) revealed three characteristics of TLCV evolution: (1) a higher nucleotide substitution rate, (2) more frequent migration among geographically isolated host populations, and (3) more frequent host changes to different plant families. While TMGMV is an RNA virus, TLCV has DNA genomes. In animal viruses, RNA viruses tend to evolve faster than DNA viruses. Our results indicated that this trend may not hold for plant viruses.     

Molecular detection of Bartonella species infecting rodents in Slovenia

Rodents, collected in three zoogeographical regions across Slovenia, were tested for the presence of bartonellae using direct PCR-based amplification of 16S/23S rRNA gene intergenic spacer region (ITS) fragments from splenic DNA extracts. Bartonella DNA was detected in four species of rodents, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius and Clethrionomys glareolus, in all three zoogeographic regions at an overall prevalence of 40.4%. The prevalence of infection varied significantly between rodent species and zoogeographical regions. Comparison of ITS sequences obtained from bartonellae revealed six sequence variants. Four of these matched the ITS sequences of the previously recognized species, Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and Bartonella birtlesii, but one was new. The identity of the bartonellae from which the novel ITS sequences was obtained were further assessed by sequence analysis of cell division protein-encoding gene (ftsZ) fragments. This analysis demonstrated that the strain is most likely a representative of possible new species within the genus.     

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.