The Experts below are selected from a list of 480 Experts worldwide ranked by ideXlab platform
Toyohiko Nishizawa - One of the best experts on this subject based on the ideXlab platform.
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Reproducibility of antigen-immobilized enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA for quantitative detection of NNV particles.
Journal of virological methods, 2019Co-Authors: Hyun Jung Gye, Toyohiko NishizawaAbstract:Nervous necrosis virus (NNV) is a fish virus belonging to family Nodaviridae. In this study, we prepared partially aggregated and monometric NNV particles to determine reproducibility of two different enzyme-linked immunosorbent assays (ELISAs): antigen-immobilized ELISA and sandwich ELISA. Passing ratios of purified NNV particles through ultrafilters with molecular weight cut off (MWCO) of 105, 3 × 105 and 106 were 0%, 35.2% and 80.3%, respectively, suggesting that purified NNV particles were partially aggregated whereas those in filtrates with MWCO of 3 × 105 could be monometric. Both NNV particles were subjected to ELISAs. Reduction ratios of ELISA values by 2-fold dilution of antigens were 50% in sandwich ELISA regardless of aggregation state of NNV particles. In contrast, those in antigen-immobilized ELISA were 42% (partially aggregated NNV) to 43% (monometric NNV), which were lower than the theoretical value (50%). This could be due to changes in aggregation state of NNV particles during dry-immobilization. Sandwich ELISA has excellent reproducibility from five times of experiments, in comparison with antigen-immobilized ELISA. Furthermore, available range of regression lines (R2 > 0.99) in sandwich ELISA was wider than that in antigen-immobilized ELISA. These results revealed that sandwich ELISA had better quantitativeness, reproducibility and available range of ELISA values than antigen-immobilized ELISA.
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Altered conformational structures of nervous necrosis virus surface protrusions and free coat proteins after incubation at moderate-low temperatures
Scientific reports, 2019Co-Authors: Hyun Jung Gye, Toyohiko NishizawaAbstract:Nervous necrosis virus (NNV) is a pathogenic fish virus belonging to family Nodaviridae. The objective of this study was to analyze stabilities of NNV surface protrusion and free coat protein (CP) conformational structures by analyzing changes of NNV infectivity and antigenicity after incubation at moderate-low temperatures. When cultured NNV suspension was incubated at 45 °C, its infectivity declined gradually but its antigenicity maintained. In contrast, both infectivity and antigenicity of purified NNV declined after incubation at 45 °C. After heat-treatment, surface protrusions of NNV particles disappeared completely, although viral particle structures maintained. Therefore, the reduction in NNV infectivity appeared to specifically occur as a result of heat-denaturation of virus surface protrusions. The loss of NNV infectivity in the presence of fetal bovine serum (FBS) was delayed compared to virus heated in the absence of FBS, demonstrating that FBS could function as a stabilizer for conformational structures of NNV surface protrusions. Moreover, the stabilizing function of FBS changed depending on salt concentration. Continued maintenance of antigenicity for heated cultured NNV suspension containing free-CPs may suggest that conformational structures corresponding to protrusion-domain of free-CP are more heat-stable than those of surface protrusions on NNV particles.
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Lack of nervous necrosis virus (NNV) neutralizing antibodies in convalescent sevenband grouper Hyporthodus septemfasciatus after NNV infection.
Vaccine, 2018Co-Authors: Hyun Jung Gye, Toyohiko NishizawaAbstract:Abstract Viral nervous necrosis (VNN) is caused by nervous necrosis viruses (NNVs) belonging to genus Betanodavirus ( Nodaviridae ). It is one of the most serious diseases in aquaculture industry worldwide. In the present study, the kinetics of NNV-infectivity and NNV-specific antibodies in convalescent sevenband grouper Hyporthodus septemfasciatus after NNV infection was determined. When fish were infected with NNV at 17.5 °C, and reared for 84 days at natural seawater temperature (increasing rate: approximately 0.1 °C/day), NNV infectivity peaked on day 14 with 10 7.80 TCID 50 /g at the highest, and declined to below the detection limit. When convalescent fish were reared at 27 °C, and re-infected with NNV at 10 4.3 or 10 6.3 TCID 50 /fish, no mortality was observed although NNV multiplied up to 10 8.80 and 10 7.80 TCID 50 /g at the highest, respectively, suggesting NNV-specific immune response. It also revealed that convalescent fish were re-infected by NNV although NNV multiplication was strongly regulated. Interestingly, NNV-specific antibodies were detectable in 20% and ≥80% of convalescent fish before and after re-infection with NNV, respectively. However, no NNV-neutralizing activity was detected before and after re-infection in almost all of the convalescent fish. Therefore, NNV-neutralizing antibodies might not be necessary for the protection of convalescent fish against NNV re-infection after previous NNV infection.
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Heat-denaturation of conformational structures on nervous necrosis virus for generating neutralization antibodies
Aquaculture, 2018Co-Authors: Hyun Jung Gye, Min-ji Park, Wi-sik Kim, Toyohiko NishizawaAbstract:Abstract Nervous necrosis virus (NNV) belongs to genus Betanodavirus within family of Nodaviridae. It is highly pathogenic to various marine fishes. In this study, we determined conformational structures of NNV epitopes for generating NNV-neutralizing antibodies using several rabbit antisera and mouse monoclonal antibodies (MAbs). Rabbit antiserum against naive NNV particles (anti-NNV serum) with high NNV-neutralization activity reacted with naive NNV particles, but not with heat-denatured NNV particles, suggesting that NNV-neutralizing antibodies in the anti-NNV serum could not react with heat-denatured NNV antigens. In contrast, rabbit antiserum against heat-denatured NNV particles (anti-HD-NNV serum) reacted with both naive and heat-denatured NNV particles. However, no NNV-neutralization activity was observed in this anti-HD-NNV serum, suggesting that heat-denatured NNV particles had no epitope for generating NNV-neutralizing antibodies. Mouse MAbs with NNV-neutralization activity also reacted with naive NNV particles, but not react with heat-denatured NNV particles. Therefore, epitopes of NNV particles for generating NNV-neutralizing antibodies had conformational structures sensitive to heat-denaturation. NNV-neutralizing antibodies were also generated by immunization with SDS-denatured NNV antigens after dialysis and ultrafiltration, suggesting that epitopes for generating NNV-neutralizing antibodies might have relatively simple conformational structures.
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Purification of nervous necrosis virus (NNV) particles by anion-exchange chromatography.
Journal of virological methods, 2016Co-Authors: Hyun Jung Gye, Toyohiko NishizawaAbstract:Nervous necrosis virus (NNV) belongs to genus Betanodavirus (family Nodaviridae). It is highly pathogenic to various marine fishes. In the present study, cultured NNV suspension was placed in dialysis tube at molecular weight cut off (MWCO) of 106 and dialyzed against Dulbecco's phosphate buffered saline (D-PBS), 15mM Tris-HCl (pH 8.0), or deionized water (DIW) for 14days followed by anion-exchange chromatography. Infectivity titers of NNV suspensions were stable during dialyses. However, the antigenicity of NNV suspension was decreased to 2.5% by D-PBS dialysis, 11.8% by Tris-HCl dialysis, and 56.2% by DIW dialysis. Anion-exchange chromatograms revealed a total of four peaks (P300, P400, P600 and P700) for NNV suspension after D-PBS dialysis. Additional two peaks (P800 and P-OH) were detected in the NNV suspension after Tris-HCl or DIW dialysis. The substance from the P700 peak had the highest NNV-infectivity. Peak P700 commonly shared by the NNV suspensions after dialysis against the three different buffers. After Tris-HCl dialysis, no other protein except NNV coat protein (CP) at Mr 41,000 was detected from P700. However, after D-PBS dialysis, the P700 peak also contained P600 antigens. Therefore, the P700 peak after Tris-HCl dialysis represented the peak of highly purified NNV particles.
Kyle L Johnson - One of the best experts on this subject based on the ideXlab platform.
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A 3′ terminal stem-loop structure in Nodamura virus RNA2 forms an essential cis-acting signal for RNA replication
Virus Research, 2010Co-Authors: John J Rosskopf, John H Upton, Lizette Rodarte, Tammy A Romero, Ming-ying Leung, Michela Taufer, Kyle L JohnsonAbstract:Abstract Nodamura virus (NoV; family Nodaviridae ) contains a bipartite positive-strand RNA genome that replicates via negative-strand intermediates. The specific structural and sequence determinants for initiation of nodavirus RNA replication have not yet been identified. For the related nodavirus Flock House virus (FHV) undefined sequences within the 3′-terminal 50 nucleotides (nt) of FHV RNA2 are essential for its replication. We previously showed that a conserved stem–loop structure (3′SL) is predicted to form near the 3′ end of the RNA2 segments of seven nodaviruses, including NoV. We hypothesized that the 3′SL structure from NoV RNA2 is an essential cis -acting element for RNA replication. To determine whether the structure can actually form within RNA2, we analyzed the secondary structure of NoV RNA2 in vitro transcripts using nuclease mapping. The resulting nuclease maps were 86% consistent with the predicted 3′SL structure, suggesting that it can form in solution. We used a well-defined reverse genetic system for launch of NoV replication in yeast cells to test the function of the 3′SL in the viral life cycle. Deletion of the nucleotides that comprise the 3′SL from a NoV2-GFP chimeric replicon resulted in a severe defect in RNA2 replication. A minimal replicon containing the 5′-terminal 17 nt and the 3′-terminal 54 nt of RNA2 (including the predicted 3′SL) retained the ability to replicate in yeast, suggesting that this region is able to direct replication of a heterologous mRNA. These data suggest that the 3′SL plays an essential role in replication of NoV RNA2. The conservation of the predicted 3′SL suggests that this common motif may play a role in RNA replication for the other members of the Nodaviridae .
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A 3' terminal stem-loop structure in Nodamura virus RNA2 forms an essential cis-acting signal for RNA replication.
Virus research, 2010Co-Authors: John J Rosskopf, John H Upton, Lizette Rodarte, Tammy A Romero, Ming-ying Leung, Michela Taufer, Kyle L JohnsonAbstract:Nodamura virus (NoV; family Nodaviridae) contains a bipartite positive-strand RNA genome that replicates via negative-strand intermediates. The specific structural and sequence determinants for initiation of nodavirus RNA replication have not yet been identified. For the related nodavirus Flock House virus (FHV) undefined sequences within the 3'-terminal 50 nucleotides (nt) of FHV RNA2 are essential for its replication. We previously showed that a conserved stem-loop structure (3'SL) is predicted to form near the 3' end of the RNA2 segments of seven nodaviruses, including NoV. We hypothesized that the 3'SL structure from NoV RNA2 is an essential cis-acting element for RNA replication. To determine whether the structure can actually form within RNA2, we analyzed the secondary structure of NoV RNA2 in vitro transcripts using nuclease mapping. The resulting nuclease maps were 86% consistent with the predicted 3'SL structure, suggesting that it can form in solution. We used a well-defined reverse genetic system for launch of NoV replication in yeast cells to test the function of the 3'SL in the viral life cycle. Deletion of the nucleotides that comprise the 3'SL from a NoV2-GFP chimeric replicon resulted in a severe defect in RNA2 replication. A minimal replicon containing the 5'-terminal 17 nt and the 3'-terminal 54 nt of RNA2 (including the predicted 3'SL) retained the ability to replicate in yeast, suggesting that this region is able to direct replication of a heterologous mRNA. These data suggest that the 3'SL plays an essential role in replication of NoV RNA2. The conservation of the predicted 3'SL suggests that this common motif may play a role in RNA replication for the other members of the Nodaviridae.
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RNAVLab: A virtual laboratory for studying RNA secondary structures based on grid computing technology
Parallel computing, 2008Co-Authors: Michela Taufer, Ming-ying Leung, Thamar Solorio, Abel Licon, David Mireles, Roberto Araiza, Kyle L JohnsonAbstract:As ribonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation, their secondary structures have been the focus of many recent studies. Despite the computing power of supercomputers, computationally predicting secondary structures with thermodynamic methods is still not feasible when the RNA molecules have long nucleotide sequences and include complex motifs such as pseudoknots. This paper presents RNAVLab (RNA Virtual Laboratory), a virtual laboratory for studying RNA secondary structures including pseudoknots that allows scientists to address this challenge. Two important case studies show the versatility and functionalities of RNAVLab. The first study quantifies its capability to rebuild longer secondary structures from motifs found in systematically sampled nucleotide segments. The extensive sampling and predictions are made feasible in a short turnaround time because of the grid technology used. The second study shows how RNAVLab allows scientists to study the viral RNA genome replication mechanisms used by members of the virus family Nodaviridae.
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Recovery of infectivity from cDNA clones of nodamura virus and identification of small nonstructural proteins.
Virology, 2003Co-Authors: Kyle L Johnson, B. Duane Price, L. Andrew BallAbstract:Nodamura virus (NoV) was the first isolated member of the Nodaviridae, and is the type species of the alphanodavirus genus. The alphanodaviruses infect insects; NoV is unique in that it can also lethally infect mammals. Nodaviruses have bipartite positive-sense RNA genomes in which RNA1 encodes the RNA-dependent RNA polymerase and the smaller genome segment, RNA2, encodes the capsid protein precursor. To facilitate the study of NoV, we generated infectious cDNA clones of its two genomic RNAs. Transcription of these NoV1 and NoV2 cDNAs in mammalian cells led to viral RNA replication, protein synthesis, and production of infectious virus. Subgenomic RNA3 was produced during RNA replication and encodes nonstructural proteins B1 and B2 in overlapping ORFs. Site-directed mutagenesis of these ORFs, followed by SDS-PAGE and MALDI-TOF mass spectrometry analyses, showed synthesis of B1 and two forms of B2 (B2-134 and B2-137) during viral replication. We also characterized a point mutation in RNA1 far upstream of the RNA3 region that resulted in decreased RNA3 synthesis and RNA2 replication, and a reduced yield of infectious particles. The ability to reproduce the entire life cycle of this unusual nodavirus from cDNA clones will facilitate further analysis of NoV RNA replication and pathogenesis.
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Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases.
Journal of General Virology, 2001Co-Authors: Karyn N. Johnson, Kyle L Johnson, Ranjit Dasgupta, Theresa Gratsch, L. Andrew BallAbstract:The Nodaviridae are a family of isometric RNA viruses that infect insects and fish. Their genomes, which are among the smallest known for animal viruses, consist of two co-encapsidated positive-sense RNA segments: RNA1 encodes the viral contribution to the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome, whereas RNA2 encodes the capsid protein precursor. In this study, the RNA1 sequences of two insect nodaviruses - Nodamura virus (the prototype of the genus) and Boolarra virus - are reported as well as detailed comparisons of their encoded RdRps with those of three other nodaviruses of insects and one of fish. Although the 5' and 3' untranslated regions did not reveal common features of RNA sequence or secondary structure, these divergent viruses showed similar genome organizations and encoded RdRps that had from 26 to 99% amino acid sequence identity. All six RdRp amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. A search for structural homologues in the protein structure database identified the poliovirus RdRp, 3D(pol), as the best template for homology modelling of the RNA polymerase domain of Pariacoto virus and allowed the construction of a congruent three-dimensional model. These results extend our understanding of the relationships among the RNA1 segments of nodaviruses and the predicted structures of their encoded RdRps.
Toshihiro Nakai - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Nodaviridae.
Journal of General Virology, 2019Co-Authors: A.s. Sahul Hameed, A. S. Ninawe, Toshihiro Nakai, Shau-chi Chi, K. L. JohnsonAbstract:The family Nodaviridae includes two genera, Alphanodavirus and Betanodavirus. The family name derives from the Japanese village of Nodamura where Nodamura virus was first isolated from Culex tritaeniorhynchus mosquitoes. Virions are non-enveloped and spherical in shape with icosahedral symmetry (T=3) and diameters ranging from 25 to 33 nm. The genome consists of two molecules of single-stranded positive-sense RNA: RNA1 and RNA2. The virion capsid consists of 180 protein subunits arranged on a T=3 surface lattice. Alphanodaviruses infect insects, whereas betanodaviruses are pathogens of fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Nodaviridae, which is available at www.ictv.global/report/Nodaviridae.
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ICTV virus taxonomy profile: Sarthroviridae.
The Journal of general virology, 2018Co-Authors: A.s. Sahul Hameed, A. S. Ninawe, Toshihiro Nakai, Shau-chi Chi, K. L. JohnsonAbstract:The family Sarthroviridae includes a single genus, Macronovirus, which in turn includes a single species, Macrobrachium satellite virus 1. Members of this species, named extra small virus, are satellite viruses of Macrobrachium rosenbergii nodavirus, an unclassified virus related to members of the family Nodaviridae. Both viruses have isometric, spherical virions, infect giant freshwater prawns and together cause white tail disease, which is responsible for mass mortalities and severe economic losses in hatcheries and farms. Infection is caused by both vertical and horizontal transmission of virus. Aquatic insects act as a carrier to transmit the disease in prawns. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Sarthroviridae, which is available at www.ictv.global/report/sarthroviridae.
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Current Knowledge on Viral Nervous Necrosis (VNN) and its Causative Betanodaviruses
Israeli Journal of Aquaculture-bamidgeh, 2009Co-Authors: Toshihiro Nakai, K. Mori, Takuma Sugaya, Toyohiro Nishioka, K Mushiake, H. YamashitaAbstract:Viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) caused by betanodaviruses (Nodaviridae) has seriously damaged global marine aquaculture since its first appearance in the late 1980s. In the past two decades, more than 100 papers have been published on the disease. Although information is still limited, we now have more knowledge on the taxonomic position and molecular characteristics of betanodaviruses, and on the diagnosis, control, and infection mechanisms of the disease. This paper briefly reviews studies on VNN and betanodaviruses.
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Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis.
Virology, 1992Co-Authors: Koh-ichiro Mori, Toshihiro Nakai, Kiyokuni Muroga, Misao Arimoto, Keiichi Mushiake, Iwao FurusawaAbstract:Abstract Spherical virus particles were purified from larval striped jack ( Pseudocaranx dentex with nervous necrosis. The virus consists of nonenveloped particles, about 25 nm in diameter, and contains two single-stranded, positive-sense RNA molecules with molecular weights of 1.01 × 10 6 Da (RNA 1) and 0.49 × 10 6 Da (RNA 2), respectively. The RNAs do not have poly(A) sequences at the 3′ terminus. Virus structural proteins consist of two proteins with molecular weights of 42 and 40 kDa. When translated into cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of the 1 a protein (100 kDa), whereas RNA 2 synthesized the 2a protein (42 kDa), which is probably the coat protein of the virus, and a polypeptide of 40 kDa which appears to be the processed form of the 42-kDa protein. Under electron microscopic observation, the virus particles were found in the tissues of the central nervous system of the affected larval striped jack. From morphorogical and biochemical properties of the virus, we identified this virus as a new member of the family of Nodaviridae and designated it striped jack nervous necrosis virus.
John E. Johnson - One of the best experts on this subject based on the ideXlab platform.
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Flock house virus: a model system for understanding non-enveloped virus entry and membrane penetration.
Current topics in microbiology and immunology, 2010Co-Authors: Amy L. Odegard, Manidipa Banerjee, John E. JohnsonAbstract:The means by which non-enveloped viruses penetrate cellular membranes during cell entry remain poorly defined. Recent findings indicate that several members of this group share a common mechanism of membrane penetration in which the virus particle undergoes programmed conformational changes, leading to capsid disassembly and release of small membrane-interacting peptides. Flock House Virus (FHV), a member of the Nodaviridae family, offers some unique advantages for studying non-enveloped virus entry. The simplicity of the FHV capsid, coupled with a robust reverse genetics system for virus expression and an abundance of structural and biochemical data, make FHV an ideal model system for such studies. Here, we review the FHV atomic structure and examine how these molecular details provide insight into the mechanism of FHV entry. In addition, recent studies of FHV entry are discussed and a current model of FHV entry and membrane penetration is presented. A complete understanding of host cell entry by this minimal system will help elucidate the mechanisms of non-enveloped virus membrane penetration in general.
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Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate
Journal of virology, 2010Co-Authors: Manidipa Banerjee, Jeffrey A. Speir, Maggie H. Kwan, Rick K. Huang, Peyman P. Aryanpur, Brian Bothner, John E. JohnsonAbstract:Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membrane-disrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.
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Structural and electrostatic characterization of pariacoto virus: implications for viral assembly.
Biopolymers, 2009Co-Authors: Batsal Devkota, Anette Schneemann, John E. Johnson, Anton S. Petrov, Sébastien Lemieux, Mustafa Burak Boz, Liang Tang, Stephen C. HarveyAbstract:We present the first all-atom model for the structure of a T = 3 virus, pariacoto virus (PaV), which is a nonenveloped, icosahedral RNA virus and a member of the Nodaviridae family. The model is an extension of the crystal structure, which reveals about 88% of the protein structure but only about 35% of the RNA structure. New modeling methods, combining coarse-grained and all-atom approaches, were required for developing the model. Evaluation of alternative models confirms our earlier observation that the polycationic N- and C-terminal tails of the capsid proteins must penetrate deeply into the core of the virus, where they stabilize the structure by neutralizing a substantial fraction of the RNA charge. This leads us to propose a model for the assembly of small icosahedral RNA viruses: nonspecific binding of the protein tails to the RNA leads to a collapse of the complex, in a fashion reminiscent of DNA condensation. The globular protein domains are excluded from the condensed phase but are tethered to it, so they accumulate in a shell around the condensed phase, where their concentration is high enough to trigger oligomerization and formation of the mature virus.
Richard Thiéry - One of the best experts on this subject based on the ideXlab platform.
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Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV) Using Mass Spectrometry
Viruses, 2015Co-Authors: Aurore Chevin, Anne-sophie Dabert-gay, Magali Ribière-chabert, Philippe Blanchard, Bruno Coutard, Richard ThiéryAbstract:Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.
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Experimental infection of the honeybee (Apis mellifera L.) with the chronic bee paralysis virus (CBPV): infectivity of naked CBPV RNAs.
Virus research, 2012Co-Authors: Aurore Chevin, Richard Thiéry, Philippe Blanchard, Frank Schurr, Magali RibièreAbstract:Abstract Chronic paralysis is an infectious and contagious disease of the honeybee ( Apis mellifera L.) and is caused by the chronic bee paralysis virus (CBPV). This disease leads to death in adult bees and is therefore a serious threat for colony health. CBPV is a positive single-stranded RNA virus and its genome is composed of two RNA segments, RNA 1 and RNA 2, 3674 nt and 2305 nt, respectively. Although CBPV shares some characteristics with viruses classified into families Nodaviridae and Tombusviridae , it has not been assigned to any viral taxa yet. The characterisation of CBPV proteins and their functions are needed to better understand the mechanisms of CBPV infection. However, since honeybee cell lines are not yet available, experimental infection of adult bees is the only method currently available to propagate the virus. With the objective of studying CBPV proteins using the viral genome, we used experimental infection in adult bees to evaluate the infectivity of naked CBPV RNAs by direct inoculation. Our results demonstrated that an injection of naked RNAs, ranging from 10 9 to 10 10 CBPV copies, caused chronic paralysis. Bees inoculated with naked RNA showed chronic paralysis signs 5 days after inoculation. Moreover, injected RNAs replicated and generated viral particles. We therefore provide an in vivo experimental model that will be useful tool for further studies by using a reverse genetics system.
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Viral encephalopathy and retinopathy of Dicentrarchus labrax and Sparus aurata farmed in Tunisia
Veterinary Research Communications, 2009Co-Authors: Nadia Chérif, Richard Thiéry, Jeannette Castric, Stephane Biacchesi, Michel Brémont, Fatma Thabti, Latifa Limem, Salah HammamiAbstract:Viruses belonging to the Nodaviridae family cause disease worldwide among a large number of species of marine fish, and have been described in all continents. In the present study, a total of 69 farmed Tunisian sea bass (Dicentrarchus labrax) and 24 sea bream (Sparus aurata) samples were tested monthly for the detection of betanodavirus. The virus was identified in both species using indirect immunofluorescence assays (IFAT) and RT-PCR. In addition sequence analysis of part of the coat protein gene indicated that both species were infected by highly related, but distinct, strains belonging to the RGNNV genotype. The sequence of the coat protein gene of several strains was identical but up to 9 different sequences were detected in a single farm. In addition, viral sequences obtained from fish that were held at lower temperature (
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Molecular characterisation and phylogenetic analysis of Chronic bee paralysis virus, a honey bee virus
Virus Research, 2008Co-Authors: Violaine Olivier, Richard Thiéry, Philippe Blanchard, Soraya Chaouch, Perrine Lallemand, Frank Schurr, Olivier Celle, Eric Dubois, Noël Tordo, Rémi HoulgatteAbstract:The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.