A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures.

BACKGROUND: Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. METHODS: To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. RESULTS: Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. CONCLUSIONS: We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

In vitro screening of selected antiviral drugs against betanodavirus.

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 µM, 10 µM, 100 µM, 200 µM and 500 µM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 µM, 10 µM and 100 µM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 µM and 100 µM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.

Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus.

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12 792 nt long and organized into seven ORFs with the gene order 3'-N-X-P-Y-M-G-L-5', which encodes the nucleocapsid, phospho, movement, matrix, glyco, and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. Cloned ORFs for each gene, except L, were used to construct a protein interaction and localization map (PILM) for this virus, which shares greater than 80 % amino acid similarity in all ORFs except X and P with the sanguinolenta strain of this species (SYDV). Protein localization patterns and interactions unique to each viral strain were identified, resulting in strain-specific PILMs. Localization of CYDV and SYDV proteins in virus-infected cells mapped subcellular loci likely to be sites of replication, morphogenesis and movement.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry.

A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. Size-exclusion HPLC analysis reduced significantly the complexity by providing a constituents profile according to size. Significantly, no sample treatment was required prior to analysis thus eliminating a potential source of artifacts and degradation. Distinct profiles were associated with influenza strains as well as with vaccines from different manufacturers. Samples analyzed over several years allowed evaluation of method performance and provided stability-indicating data relating to the structural integrity of separated components. Collected chromatographic peaks were identified by gel electrophoresis and MALDI/MS of tryptic digests from excised gel bands. The challenge in obtaining high quality analytical data from complex mixtures clearly demonstrated the value of separation steps prior to MS identification. The method presented here is not intended to replace existing methodology; it is intended to provide a product specific profile to be used as a rapid screen for manufacturer, year (for annual influenza vaccines), stability or counterfeit product. It is a new screening method that provides a rapid and robust indication of products which require further investigation as a result of a deviation in their characteristic profile. Until now this tool did not exist.

[Establishment of bioassay method for antivirus potency of radix isatidis based on chemical fluorometric determination].

In the present study, the in vitro inhibitory activity of extracts from radix isatidis on neuraminidase (NA) was investigated by the chemical fluoremetic determination to establish the quality control method for antivirus action of radix isatidis. The initial study indicated that radix isatidis had obvious in vitro inhibitory activity on NA with IC50 = (0.90 +/- 0.20) mg (herb) x mL(-1). The correlation between logarithmic dose and reaction rate showed a "S" shape and a linear curve (linear equation, y = 8.7259 + 1.2169 x log(D), R = 0.9992) when the reaction rate was converted to probit-quite similar to Tamiflu's reaction curve, which hinted that radix isatidis had the same inhibitory function on NA as Tamiflu. According to the reaction type and the regularity of "parallel lines of qualitative effect", the experimental condition was optimized and a statistic method was confirmed based on the principle of bioassay statistic. Then the bioassay method for antivirus potency of radix isatidis based on fluorometric determination was established. The results of bio-potency assay showed the qualitative differences of radix isatidis samples sensitively and quantitatively. Meanwhile, this method has good reproducibility with RSD = 5.78% and reliability. The quality bioassay control method based on chemical fluorometric determination can reflect the pharmaco-dynamic features of Chinese medicine herb.

The preclinical testing of a formaldehyde inactivated Ross River virus vaccine designed for use in humans.

Ross River virus was grown in industrial facilities in vaccine-certified Vero cells in the absence of serum, inactivated using standard formalin-inactivation protocols, treated with Benzonase to digest host cell DNA and purified on a sucrose gradient. Mice given two subcutaneous injections of 0.625 microg of this vaccine or two doses of 0.156 microg vaccine with aluminium hydroxide adjuvant failed to develop a detectable viraemia after intravenous challenge with 10(6)TCID50 of the prototype strain of Ross River virus (T48). Guinea pigs immunised with one or two10 microg doses of vaccine with adjuvant also failed to develop a detectable viraemia following a similar challenge. The levels of neutralising antibody (neutralisation index 1.9-3.1) in the mice protected against challenge with 10(6)TCID50 Ross River virus were similar to those in 16 former epidemic polyarthritis patients (1.1-3.5) who had not experienced a second clinical infection with Ross River virus in the 20 years following their initial infection.

Beet necrotic yellow vein virus accumulates inside resting spores and zoosporangia of its vector Polymyxa betae BNYVV infects P. betae.

BACKGROUND: Plasmodiophorids and chytrids are zoosporic parasites of algae and land plant and are distributed worldwide. There are 35 species belonging to the order Plasmodiophorales and three species, Polymyxa betae, P. graminis, and Spongospora subterranea, are plant viral vectors. Plasmodiophorid transmitted viruses are positive strand RNA viruses belonging to five genera. Beet necrotic yellow vein virus (BNYVV) and its vector, P. betae, are the causal agents for rhizomania. RESULTS: Evidence of BNYVV replication and movement proteins associating with P. betae resting spores was initially obtained using immunofluorescence labeling and well characterized antisera to each of the BNYVV proteins. Root cross sections were further examined using immunogold labeling and electron microscopy. BNYVV proteins translated from each of the four genomic and subgenomic RNAs accumulate inside P. betae resting spores and zoospores. Statistical analysis was used to determine if immunolabelling detected viral proteins in specific subcellular domains and at a level greater than in control samples. CONCLUSION: Virus-like particles were detected in zoosporangia. Association of BNYVV replication and movement proteins with sporangial and sporogenic stages of P. betae suggest that BNYVV resides inside its vector during more than one life cycle stage. These data suggest that P. betae might be a host as well as a vector for BNYVV.

Development and evaluation of a multiplex RT-PCR for detecting main viruses and a viroid of potato.

A multiplex RT-PCR (mRT-PCR) for detecting four potato viruses (Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), and Potato leaf roll virus (PLRV)) and one potato viroid (Potato spindle tuber viroid (PSTVd)) was developed. The mRT-PCR consisted of one reaction with specific primers designed according to the sequences of coat protein (CP) genes of respective viruses and the sequence of the viroid. The entire procedure from tissue grinding to RT-PCR results takes about 4 hrs. The reliability of the method was tested on leaves sampled from the field, greenhouse and in vitro-grown plants by comparison with double-antibody sandwich ELISA (DAS-ELISA). A high correlation between these two methods was observed. The mRT-PCR was also evaluated by testing infected samples obtained from the International Potato Center, Lima, Peru; this testing confirmed its high reliability and sensitivity.

Identification of selective inhibitors of VHSV from biased combinatorial libraries of N,N'-disubstituted 2,5-piperazinediones.

A combinatorial strategy has been used to design and identify inhibitors of viral haemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus of economic importance. Two libraries of N,N'-disubstituted 2,5-piperazinediones (DKP), DKP-I and DKP-II were screened for inhibition of VHSV infectivity. Among the 98 DKP-derivatives (R1-DKP-R2) screened, a novel class of VHSV in vitro inhibitors was identified. Evidences are presented showing that the selected DKP-derivatives cause dose-dependent inhibition of VHSV infectivity in the absence of cellular toxicity. Preliminary characterization of its inhibition mechanism ruled out direct inactivation of the virus (virucidal effect) or interference with early viral replication steps. Furthermore, analysis of infection foci sizes, virus titers, viral protein accumulation and presence of cell free virus derived from VHSV-infected cell cultures in the presence of DKP-derivates suggested that virus assembly/release was impaired leading to a reduced virus spread in cell culture. New DKP-derivatives with a significant higher specific activity need to be developed to start testing its possible practical use but the selected DKP-derivatives described here may contribute to their further development as well as being tools to improve our knowledge on the fish rhabdovirus infection cycle.

Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease.

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations (K(i) = 4 microM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC(50) values ranging from 7.6 to 84.5 microM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.

The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated Brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity.

Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally diverse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.

Inhibition of potato virus Y NIa activity: preparation of monoclonal antibody directed against PVY NI protein that inhibits cleavage of PVY polyprotein.

A partially purified nuclear inclusion (NI) fraction was obtained from tobacco plants infected by potato virus Y (PVY). Four monoclonal antibodies (MAbs) were produced and characterized using this semipurified fraction as antigen. Data showed that only one was directed against NIa whereas two were directed against cytoplasmic inclusion (CI) protein and the last one against coat protein (CP). These results were due to the fact that the semipurified NI fraction was usually contaminated with CI and CP proteins. When used on in situ immunofluorescence method the anti-NIa MAb showed accumulation of the NIa protein in both nucleus and cytoplasm. In vivo, this MAb was able to detect different forms of the NIa protein including precursors and cleavage products. It was also able to inhibit the cleavage of the polyprotein detected in the semipurified NI.

Effect of genomic and subgenomic leader sequences of potato leafroll virus on gene expression.

The effect of the genomic and subgenomic leader sequence of potato leafroll polerovirus on the efficiency of translation of the downstream located genes has been studied. The results obtained in vitro and in vivo indicate that neither leader sequence functions as translational enhancer, a generally important feature of leader sequences. Deletion analyses demonstrated that both leader sequences not only decrease translation of the downstream located genes but also alter the ratio of the synthesized proteins. A correlation between the in vitro and in vivo results can be established in the case of the subgenomic leader sequence.

Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully.

Localization of the P1 protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells.

The N-terminal P1 proteinase of potato virus Y (ordinary strain group isolate PVY-O) was expressed in E. coli. Antiserum was raised against the expressed protein and used to detect the viral proteins in infected tobacco leaf tissue by Western blotting and by electron microscopy with immunogold labelling. In the immunogold localization studies P1 protein was detected in association with the cytoplasmic inclusion bodies characteristic of PVY infections and in the cytoplasm of the infected plant cells. No significant P1 antibody binding with other plant cell organelles, or with the cell wall and plasmodesmata, was detected by immunogold labelling.

In situ localization of the putative movement protein (pr17) from potato leafroll luteovirus (PLRV) in infected and transgenic potato plants.

The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.

Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase.

HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.

Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato.

Open reading frame 4 (ORF4) of the potato virus X (PVX) genome encodes an 8K protein which is a part of the "triple gene block" and is known to play a role in the cell-to-cell movement of the virus in infected plants. To locate the 8K protein and further elucidate the mechanism of cell-to-cell transport of PVX, antibodies were raised against the 8K protein and used to localize this protein in PVX-infected tobacco and in transgenic potato plants expressing the 8K protein both by subcellular fractionation and by immunolabeling with colloidal gold. The results indicated that the 8K protein was localized to the cell wall.