Presence Of Virus-like Particles Research Articles

Virus-like Particles: Measures and Biological Functions.

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.

Histological and Ultrastructural Description of Benign Adipocytic Tumors in Farmed Striped Sea Bream (Lythognathus mormyrus).

Simple SummaryMesenchymal neoplasms of the skin are common tumors in domestic animals and often reported in fish pathology. Among them, lipomas are frequently observed, but the definition of their different histological features and their classification in fish are still in their primordial stages. In the present work, the authors describe different type of lipomas found in a wild stock of striped seabream and outline their key differential microscopical features. Two new types of lipoma never observed in fish pathology, defined as spindle cell lipoma and atypical spindle cell-like lipoma, are reported.Cutaneous neoplasms affecting wild striped bream (Lythognathus mormyrus) have been recorded after their introduction in a marine aquaculture farm in the Adriatic Sea. The tumors were evident on 24% of the reared fish, showing single or multiple nodules, with a diameter ranging between 0.5–4.0 cm. Histologically, all the neoplastic lesions were located in the stratum spongiosum of the dermis and were surrounded by a thin capsule of connective tissue. The tumors were predominantly composed of adipocytes grouped and surrounded by a thin net of fibroblasts and collagen fibers. In some lipomas a mixture of adipocytes and uniform spindle cells were also observed. Fibroblasts and collagen fibers, or spindle cells, showing few mitotic figures were mainly observed in other nodules. Three of the tumors showed bands of cells with elongated nuclei. Five neoplasms differed from the classic spindle cell lipoma due to the presence of scattered giant cells. These cells presented acidophilic abundant cytoplasm with multiple hyperchromatic nuclei showing a concentric “floret-like” arrangement. The tumors were further characterized by ultrastructural observations that allowed ruling out the presence of virus-like particles within the lesions. Histological features of the masses lead to the identification of four prevalent patterns of neoplasms: lipoma, fibrolipoma, spindle cell lipoma (SCL), and atypical spindle cell-like lipoma (ASCL). The different neoplasms could arise from the transformation of mesenchymal cells of dermal origin. To the author’s knowledge, this is the first report describing key differential histological and ultrastructural features of these neoplasms in striped sea bream.

Effect of the Presence of Virus-like Particles on Bacterial Growth in Sunlit Surface and Dark Deep Ocean Environments in the Southern East China Sea

Virus-like particles (VLPs) are thought to increase the dissolved organic carbon by releasing the contents of the host cell, which, in turn, can affect bacterial growth in natural aquatic environments. Yet, experimental tests have shown that the effect of VLPs on the bacterial growth rate at different depths has seldom been studied. Bacteria–VLP interaction and the effect of VLPs on bacterial growth rate in the sunlit surface (3 m) and dark, deep ocean (130 m) environments were first explored at a test site in the southern East China Sea of the northwest Pacific. Our experimental results indicated that bacterial and virus-like particle (VLP) abundance decreased with depth from 0.8 ± 0.3 × 105 cells mL−1 and 1.8 ± 0.4 × 106 VLPs mL−1 at 3 m to 0.4 ± 0.1 × 105 cells mL−1 and 1.4 ± 0.3 × 106 VLPs mL−1 at 130 m. We found that the abundance of VLPs to Bacteria Ratio (VBR) in the dark deep ocean (VBR = 35.0 ± 5.6) was higher than in the sunlit surface environment (VBR = 22.5 ± 2.1). The most interesting finding is that in the dark, deep ocean region the bacterial growth rate in the presence of VLPs was higher (0.05 h−1) than that in virus-diluted treatments (0.01 h−1). However, there was no significant difference in the bacterial growth rates between the treatments in the sunlit surface ocean region. Deep-sea ecosystems are dark and extreme environments that lack primary photosynthetic production, and our estimates imply that the contribution of recycled carbon by viral lysis is highly significant for bacterial growth in the dark, deep ocean environment. Further work for more study sites is needed to identify the relationship of VLPs and their hosts to enable us to understand the role of VLPs at different depths in the East China Sea.

Dry Formulation of Virus-Like Particles in Electrospun Nanofibers.

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w/v) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

Kidney Involvement in COVID-19: Need for Better Definitions

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus outbreak in China at the end of 2019, increasing interest emerged regarding renal involvement during coronavirus disease 2019 (COVID-19). AKI with or without proteinuria is described in a variable percentage of patients with COVID-19, and several reports outlined an increased mortality risk in those with AKI. The mechanisms of renal injury during COVID-19 are difficult to study due to the interference of several coexisting factors, such as polypharmacy, hypoxia, and cytokine storm. Just like severe acute respiratory syndrome coronavirus, SARS-CoV-2 virus entry into target cells is facilitated by the presence of angiotensin-converting enzyme 2 (ACE2) expressed in respiratory cells as well as renal tubular cells and podocytes, making the hypothesis of a direct renal infection particularly intriguing. Farkash et al.1 observed renal tubular vacuolization and virus-like inclusions in tubular cells by electron microscopy (EM) in the autopsy of a patient with COVID-19. Similar EM findings together with suggestive immunohistochemical stain for SARS-CoV-2 proteins in tubules were shown in a few patients from two independent Chinese COVID-19 autopsy cohorts (Diao B, Wang C, Wang R, Feng Z, Tan Y, Wang H, et al. [2020] Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infection. 10.1101/2020.03.04.20031120).2 However, the same immunohistochemical was not considered specific when used by others.3 Of note, when specific and highly sensitive molecular methods for tissue viral identification were used (i.e., RNAscope3 or real-time PCR,4 the presence of SARS-CoV-2 RNA could not be demonstrated within renal tissue, and none of the aforementioned autopsy studies included viral RNA isolation in the kidney. Interestingly, viral nephropathies with direct tubular infection and viral replication within epithelial cells (i.e., BK virus nephropathy) are usually associated with substantial viruria due to the direct release of viral particles in the tubular lumen following cytopathic cell injury. So far, there is no solid evidence of a correlation between urinary levels of SARS-CoV-2 viral copies and the degree of kidney injury in patients with COVID-19.5 Moreover, the presence of viral particles by pure morphologic evaluation (EM) does not demonstrate by itself a direct cytopathic effect nor a replicative potential because it only supports viral entry. More specific morphologic correlates of viral cytopathy (nuclear atypia/dysmorphology and syncytia) have not been shown yet. Three cases of collapsing glomerulopathy during SARS-CoV-2 infection have been reported,6 presenting with severe kidney injury and variable proteinuria: in genetically predisposed subjects, increased levels of cytokines (i.e., IFNs) in patients with COVID-19 are thought to drive podocyte injury, resulting in collapsing glomerulopathy as can be observed in other viral-associated nephropathies. Although in one case, the presence of virus-like particles in podocytes was observed by EM, the hypothesized mechanism of injury does not require direct kidney infection. Taken together, these observations suggest that caution should be used when addressing the etiology of kidney dysfunction in patients with COVID-19 because unequivocal evidence of direct viral infection of the kidney is still lacking in these severe and acutely ill patients. Disclosures All authors have nothing to disclose. Funding None.

Evidence for microbes in early Neoproterozoic stromatolites

The biogenesis of stromatolites has long been discussed since many lack convincing evidence of their biological origin. This has particularly been the case with older Precambrian examples where widespread diagenetic and metamorphic processes have commonly destroyed microbial relics; in some cases however, evidence has been preserved through very early mineralisation. Precambrian stromatolites are relatively abundant but it is extremely rare that they contain calcified or dolomitized cyanobacteria and biofilms. This study provides evidence from early Neoproterozoic columnar stromatolites in the Jiuliqiao Formation from the southeastern margin of the North China Platform for fossilized extracellular polymeric substances (EPS) and cyanobacteria, along with nanospheres which may represent permineralised viruses or viral-like particles. A similar chemical composition and colour along with their embedded and cross-cutting relationships with the matrix suggest they are syn-depositional products, and not modern artefacts or the result of sample preparation. The interweaving of cyanobacterial filaments with detrital grains such as quartz and intraclasts indicates trapping, baffling and binding processes. The presence of virus-like particles and mineralized EPS with anhedral, fine-grained calcite suggests they may have provided initial nucleation sites for subsequent carbonate precipitation. This study demonstrates the use of a high-definition scanning electron microscope to detect fossilized (calcified) filamentous and spheroidal bacteriomorphs within ancient stromatolites in order to confirm their biogenesis.

Desulfovibrio alaskensis prophages and their possible involvement in the horizontal transfer of genes by outer membrane vesicles

Desulfovibrio alaskensis is a Gram-negative bacterial species that belongs to the group of Sulphate Reducing Bacteria (SRB) and presents prophages in genomes, a common characteristic of the genus Desulfovibrio. Genetic material can be transported by outer membrane vesicles, however, no data regarding the production of these vesicles has been reported for D. alaskensis. To verify the expression of D. alaskensis prophages and their involvement with outer membrane vesicles, the DSM16109 strain was used. The DSM16109 strain had three prophages and presented reduced growth after mitomycin C addition when compared to the control culture. This reduction was accompanied by the presence of virus-like particles (VLPs), indicating mitomycin C dependent prophage induction. The increase in the number of cap gene copies and transcriptions of the three prophages was verified in the control sample, however, without the formation of VLPs. Prophage genes were identified in outer membrane vesicles from cultures treated and not treated with mitomycin C. DSM16109 prophages are expressed spontaneously but only in the presence of mitomycin C was it possible to observe VLP formation. Due to the genetic material detection from the prophages within outer membrane vesicles, this property may be related to the horizontal transfer of viral genes.

The psorosis disease of citrus: a pale light at the end of the tunnel

Abstract First reported in 1896, psorosis was the first citrus disease proven to be graft transmissible and also the first for which eradication and budwood certification programs were launched to prevent its economic damage. For many years psorosis etiology remained elusive and only in 1986 it was associated with the presence of virus-like particles in infected plants. However, in the last two decades a virus with unusual morphology (Citrus psorosis virus, CPsV) was characterized and closely associated with psorosis disease as previously defined by field symptoms and by biological indexing in sensitive indicator plants. With a tripartite, negative-sense, RNA genome and a ~48 kDa coat protein, CPsV, the presumed causal agent of psorosis, is the type member of the genus Ophiovirus, within the new family Ophioviridae. Availability of the complete genomic sequence of two CPsV isolates and partial sequences of many others has enabled i) setting up rapid and sensitive RNA-based detection methods, ii) testing different citrus and relatives for resistance to CPsV, iii) identification of the two components (psorosis A and psorosis B) traditionally associated with non-scaled and scaled bark inoculum, respectively, from psorosis-infected plants and study their interactions, iv) analysis of genetic variation and evolutionary forces shaping the CPsV populations, v) preliminary studies on the interactions between virus and host factors and vi) development of transgenic citrus plants expressing variable degrees of resistance to CPsV. In summary, 120 years after the first report on psorosis we start seeing a pale light at the end of the tunnel.

Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous.

Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9-1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.

Human endogenous retrovirus W activity in cartilage of osteoarthritis patients.

The etiology of viruses in osteoarthritis remains controversial because the prevalence of viral nucleic acid sequences in peripheral blood or synovial fluid from osteoarthritis patients and that in healthy control subjects are similar. Until now the presence of virus has not been analyzed in cartilage. We screened cartilage and chondrocytes from advanced and non-/early osteoarthritis patients for parvovirus B19, herpes simplex virus-1, Epstein Barr virus, cytomegalovirus, human herpes virus-6, hepatitis C virus, and human endogenous retroviruses transcripts. Endogenous retroviruses transcripts, but none of the other viruses, were detected in 15 out the 17 patients. Sequencing identified the virus as HERV-WE1 and E2. HERV-W activity was confirmed by high expression levels of syncytin, dsRNA, virus budding, and the presence of virus-like particles in all advanced osteoarthritis cartilages examined. Low levels of HERV-WE1, but not E2 envelope RNA, were observed in 3 out of 8 non-/early osteoarthritis patients, while only 3 out of 7 chondrocytes cultures displayed low levels of syncytin, and just one was positive for virus-like particles. This study demonstrates for the first time activation of HERV-W in cartilage of osteoarthritis patients; however, a causative role for HERV-W in development or deterioration of the disease remains to be proven.

GagPol-specific CD4+T-cells increase the antibody response to Env by intrastructural help

BackgroundImmunization of rhesus macaques against Gag of SIV resulted in a more rapid appearance of Env antibodies after infection with SIV or SHIV challenge viruses although the vaccines lacked an Env component. We therefore explored whether T helper cells specific for internal HIV proteins could provide intrastructural help for Env-specific B cells and thus increase the Env antibody response.ResultsMice were immunized by adenoviral vector or DNA vaccines against GagPol and then boosted with virus-like particles (VLP) containing GagPol and Env. Env-specific antibody levels after the VLP booster immunizations were significantly higher in GagPol-immunized mice than in mock-vaccinated controls. Adoptive transfer of CD4+ T cells from GagPol-immunized mice also enhanced the Env antibody response to VLP immunization in the recipient mice. Depending on the presence of VLPs, co-cultivation of CD4+ T cells from GagPol-primed mice with BCR transgenic B cells specific for a protein presented on the surface of the VLPs also resulted in the activation of the B and T cells.ConclusionsOur study indicates that GagPol-specific T helper cells may provide intrastructural help for Env antibody responses. This cross-talk between immune responses directed against different components of the retroviral particle may be relevant for the immunopathogenesis of retroviral infections and allow to improve virus like particle vaccine approaches against HIV.

First Report of a Novel Potyvirus from Florida Causing Chlorotic Mottling in Squash (Cucurbita pepo).

During the 2010 to 2011 growing seasons, nine cucurbit leaf samples including cantaloupe, cucumber, pumpkin, squash, and watermelon, showing mosaic and mottling, were collected from fields in the Homestead and Tampa areas in Florida (1). Eight of the nine samples were positive by dot-immunobinding assay (DIBA) and reverse transcription (RT)-PCR for either Watermelon mosaic virus (WMV), Papaya ringspot virus (PRSV-W), or mixed infection of both viruses. One squash sample from the Homestead area showing unique symptoms including chlorotic spots, yellowing, mottling, vein clearing, and mild mosaic was negative by RT-PCR against PRSV-W, Squash vein yellowing virus (SqVYV), WMV, and Zucchini yellow mosaic virus (ZYMV).The presence of virus-like particles (VLP) from symptomatic squash leaves (1) was prepared as described previously (2). Typical potyvirus-like particles ~700 nm long and 12 to 14 nm wide were observed by electron microscope from VLP preparations. Analysis of VLP on SDS-PAGE demonstrated a slightly larger coat protein (CP) (37 kDa compared with PRSV-W [35 kDa]). Sap from symptomatic squash leaf samples or VLP was mechanically inoculated to 10 squash seedlings at cotyledon stage using 0.1 M K2HPO4 buffer. Chlorotic spots were observed on the first true leaf 7 days post inoculation. However, symptoms became more severe by 2 to 3 weeks post inoculation and systemically infected leaves showed chlorosis and mottling similar to the original symptoms when tissues were collected from the field. Mock-inoculated control squash seedlings did not produce any symptoms. Symptomatic leaves from mechanically infected squash plants were used for VLP preparations and virus particles and size of the CP on SDS-PAGE was observed as before. Total RNA was extracted from VLP (2) and tested by RT-PCR using universal Potyviridae primers (forward primer 5'-CACGGATCCCGGG (T)17AGC and reverse primer 5'-GGBAAYAAYAGYGGDCARCC (3) to amplify a fragment from the 3' end of the genome (including part of NIb gene, whole CP). A band of 1.2 kb was observed when the PCR product was analyzed on 1% agarose gel. PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, USA), cloned (pGEM-T Easy Vector, Promega, USA), and sequenced in both directions. Consensus sequence was obtained from at least five clones and submitted to GenBank (KC522968). A BLASTn comparing the sequence from the squash potyvirus to others in GenBank found the highest similarity was 72.0% at nucleotide level and 64.8% at amino acid level with PRSV-W (JN831646), and less than 70% nucleotide similarity with WMV (NC_006262) and SqVYV (NC_010521). Based on the particle morphology, CP size on SDS-PAGE, nucleotide identity with other cucurbit potyviruses, and unique symptoms, it is concluded that this could be a new potyvirus. The threshold for classifying distinct species in Potyviridae is less than 76% identity at nucleotide level for either CP gene or the whole genome (4). This virus has been tentatively named as Squash chlorosis mottling virus (SqCMV). Florida is one of the leading states in acreage and production of cucurbits in the United States. The emergence of this new virus could be a potential future threat to cucurbits production.

Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis

A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen.

Feline leukaemia virus: Half a century since its discovery

In the early 1960s, Professor William (Bill) F.H. Jarrett was presented with a time-space cluster of cats with lymphoma identified by a local veterinary practitioner, Harry Pfaff, and carried out experiments to find if the condition might be caused by a virus, similar to lymphomas noted previously in poultry and mice. In 1964, the transmission of lymphoma in cats and the presence of virus-like particles that resembled 'the virus of murine leukaemias' in the induced tumours were reported in Nature. These seminal studies initiated research on feline leukaemia virus (FeLV) and launched the field of feline retrovirology. This review article considers the way in which some of the key early observations made by Bill Jarrett and his coworkers have developed in subsequent years and discusses progress that has been made in the field since FeLV was first discovered.

Targeting Norovirus Infection—Multivalent Entry Inhibitor Design Based on NMR Experiments

Noroviruses attach to their host cells through histo blood group antigens (HBGAs), and compounds that interfere with this interaction are likely to be of therapeutic or diagnostic interest. It is shown that NMR binding studies can simultaneously identify and differentiate the site for binding HBGA ligands and complementary ligands from a large compound library, thereby facilitating the design of potent heterobifunctional ligands. Saturation transfer difference (STD) NMR experiments, spin-lock filtered NMR experiments, and interligand NOE (ILOE) experiments in the presence of virus-like particles (VLPs), identified compounds that bind to the HBGA binding site of human norovirus. Based on these data two multivalent prototype entry-inhibitors against norovirus infection were synthesized. A surface plasmon resonance based inhibition assay showed avidity gains of 1000 and one million fold over a millimolar univalent ligand. This suggests that further rational design of multivalent inhibitors based on our strategy will identify potent entry-inhibitors against norovirus infections.

Some Genetic, Biochemical and Morphological Analysis of Selected Powdery Mildew Strains at the Beginning of Sporulation on Barley

The present work analyzes some characteristics of four powdery mildew pathotypes, RU-3, Sk-5/11, Sk-12/1 and A-4/0, selected from the wild strains of BGH from Central European regions. Our results showed that the studied BGH strains differ in the virulence and avirulence genes in their genomes, in prolongation of their asexual phase of the growth and also in morphological and biochemical characteristics. Protein analysis confirmed the genetic differences between the studied powdery mildew pathotypes. Abundant acid glucanases in all studied BHG pathotypes were found between molecular weights Mr ? 25-35 kDa and 11-22kDa. Races RU-3 and A-4/0 also contained low molecular weight glucanases with Mr ? 9-14kDa. Immunological analyses showed higher specificity of pathogen chitinases to plant antibody compared to barley cultivars carrying different dominant/semidominant resistance genes. Rabbit antibody prepared against the plant interacellular acid chitinase Chi 14.4 (PR-4) gave the positive signal for two powdery mildew races, Sk-5/11 and A-4/0. These pathotypes were more aggressive compared to races Sk-12/1 and RU-3. Their genomes contained more virulence genes and asexual phase of the growth was shorter. Ultrastructural analyses of BGH body in the sensitive barley cultivar cells, showed presence of virus like particles, which probably play role by the synthesis of some PR-proteins with hydrolytic function. Genetic and biochemical analyses indicate that some powdery mildew pathotypes contain genes in their genome which are orthological to those in their hosts, which makes them suitable subjects for the future as a source of new resistance genes for plant breeding.

Canarypox and fowlpox viruses as recombinant vaccine vectors: an ultrastructural comparative analysis

Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox virus (FP) represent efficient and safe vaccine vectors, as they correctly express transgenes in human cells, elicit complete immune responses, and show protective efficacy in preclinical animal models. At present, no information is available on the differences in the abortive replication of these two avipox viruses in mammalian cells. In the present study, the replicative cycles of CP and FP, wild-type and recombinants, are compared in permissive and non-permissive cells, using transmission electron microscopy. We demonstrate that in non-permissive cells, the replicative cycle is more advanced in FP than in CP, that human cells, whether immune or not, are less permissive to avipox replication than monkey cells, and that the presence of virus-like particles only occurs after FP infection. Overall, these data suggest that the use of FP recombinants is more appropriate than the use of CP for eliciting an immune response.

ULTRASTRUCTURAL CHARACTERIZATION OF THE LYTIC CYCLE OF AN INTRANUCLEAR VIRUS INFECTING THE DIATOM CHAETOCEROS CF. WIGHAMII(BACILLARIOPHYCEAE) FROM CHESAPEAKE BAY, USA(1).

Numerous microalgal species are infected by viruses that have the potential to control phytoplankton dynamics by reducing host populations, preventing bloom formation, or causing the collapse of blooms. Here we describe a virus infecting the diatom Chaetoceros cf. wighamii Brightw. from the Chesapeake Bay. To characterize the morphology and lytic cycle of this virus, we conducted a time-course experiment, sampling every 4 h over 72 h following viral inoculation. In vivo fluorescence began to decline 16 h after inoculation and was reduced to <19% of control cultures by the end of experiment. TEM confirmed infection within the first 8 h of inoculation, as indicated by the presence of virus-like particles (VLP) in the nuclei. VLP were present in two different arrangements: rod-like structures that appeared in cross-section as paracrystalline arrays of hexagonal-shaped profiles measuring 12 ± 2 nm in diameter and uniformly electron-dense hexagonal-shaped particles measuring ∼ 22-28 nm in diameter. Nuclei containing paracrystalline arrays were most prevalent early in the infection cycle, while cells containing VLP increased and then declined toward the end of the cycle. The proportion of nuclei containing both paracrystalline arrays and VLP remained relatively constant. This pattern suggests that rod-like paracrystalline arrays fragmented to produce icosahedral VLP. C. cf. wighamii nuclear inclusion virus (CwNIV) is characterized by a high burst size (averaged 26,400 viruses per infected cell) and fast generation time that could have ecological implications on C. cf. wighamii population control.

Mycoviruses of filamentous fungi and their relevance to plant pathology

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal-virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.

Alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (BVDV)

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro. The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (10(5.16) TCID(50)/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24-48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV-FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).After microinjection of BVDV under the ZP, significantly more (p<0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV-FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos. These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.