Three-dimensional structure of the Chinese Sacbrood bee virus

The RNA of Chinese Sacbrood Bee Virus (CSBV) was purified and used as template to obtain a 1096 bp cDNA fragment by RT-PCR amplification. This DNA fragment was cloned into pGEM-T Easy Vector for sequencing. Analyses of the sequenced CSBV RNA fragment revealed a nucleotide sequence homology of 87.6% and a deduced amino-acid sequence homology of 94.6% with that of the Sacbrood Virus (SBV), indicating that CSBV is a different but highly homologous virus of SBV. The three-dimensional (3D) structure of CSBV was determined at 2.5 nm resolution by using electron cryo-microscopy (cryoEM) and computer reconstruction methods. The 3-D structure showed that the capsid has a T= 1 (or P= 3) icosahedral capsid shell with a smooth surface. There were 12 pentons at its icosahedral vertices (5-fold axes) and 132 holes penetrating the shell. The 3-D structure also revealed densities corresponding to the CSBV genome, suggesting icosahedrally-ordered RNA organization, a novel feature not previously reported for any picornavi     

New insights of Sacbrood virus

正Over the last several years,major efforts have been expended to study viral infection of honeybees mainly due to colony losses around the world(Allen M,et al.,1996).It seems that honeybees are infected with numerous viruses mounting to 18so far.Infection may be asymptomatic but could still have adverse effects on the bee and may even cause death resulting in colony collapse.Sacbrood virus(SBV)is the most widely distributed of all honey bee viruses.     

中蜂囊状幼虫病毒RdRp基因dsRNA干扰的研究

中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)可以引起中蜂幼虫死亡,给中蜂的养殖造成严重的威胁。其RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)是病毒复制中必不可少的中心酶,控制着病毒的复制和翻译过程。本研究以CSBV RdRp基因为靶标,选取两个干扰区域RdRp-1和RdRp-2,并构建相应的dsRNA表达载体,获取dsRNA后进行RNAi实验,通过qRT-PCR检测CSBV RdRp基因的表达情况。实验结果显示:干扰片段RdRp-1不能显著下调RdRp基因的转录,而干扰片段RdRp-2可显著性下调RdRp表达并具有剂量依赖性,当添食2μg dsRdRp-2时,在72 h RdRp基因表达下调了85%,CSBV的衣壳蛋白VP1基因下调表达78%,幼虫死亡率降低60%,表明RdRp基因可以作为RNA干扰的靶标用于CSBV防治,本研究为后期在养蜂场进行蜜蜂病毒病的防治奠定了基础。     

Three-dimensional structure of the inner core of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled icosahedral virus.Using electron cryomicroscopy and computer reconstruction techniques, we have determined a 3.3 nm resolution three-dimensional (3D) structure of the inner shell capsid without the outer shell and viral RNA. The results show that the inner shell is a thin, densely packed, smooth structure, which provides a scaffold for the full virus. A total of 120 copies of the major inner shell capsid protein P3 forms 60 dimers arranged in a T=1 icosahedral lattice. A close examination on the subunit packing of the T=1 inner core P3 with that of the T=13l outer shell P8 indicated that P8 trimers connect with P3 through completely non-equivalent, yet highly specific, intermolecular interactions.     

Three-dimensional structure of the inner core of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled icosahedral virus. Using electron cryomicro-scopy and computer reconstruction techniques, we have determined a 3.3 nm resolution three-dimensional (3D) structure of the inner shell capsid without the outer shell and viral RNA. The results show that the inner shell is a thin, densely packed, smooth structure, which provides a scaffold for the full virus. A total of 120 copies of the major inner shell capsid protein P3 forms 60 dimers arranged in a T=1 icosahedral lattice. A close examination on the subunit packing of the T=1 inner core P3 with that of the T=13/ outer shell P8 indicated that P8 trimers connect with P3 through completely non-equivalent, yet highly specific, intermolecular interactions.     

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.     

A filamentous virus of the honey bee

A common and widespread disease of honey bees, Apis mellifera, is caused by an unoccluded, Feulgen-positive, filamentous nuclear virus. Ovoid viral particles seen in diseased bee hemolymph consisted of a folded nucleocapsid within a viral envelope and were 0.40 by 0.10 μm. Virions with unfolded nucleocapsids were about 3060 by 60 nm. The disease was transmissible to bees both per os and by injection, but efforts to infect oriental cockroaches, Blatta orientalis, and the greater wax moth, Galleria mellonella, failed. The disease is apparently the same as that described as a rickettsial disease of European bees.     

Transpuparial transmission of Kashmir bee virus and sacbrood virus in the honey bee (Apis mellifera)

When particles of Kashmir bee virus (KBV) and sacbrood virus (SBV) were fed to larvae of the honey bee, Apis mellifera, in Australian colonies, the resulting pupae became in apparently infected. There was no statistically significant difference in the susceptibility of 1, 2, 3 or 4-day-old larvae for either virus, but 5-day-old larvae were significantly less susceptible to SBV than younger larvae. There was no significant difference in the proportions of pupae that became in apparently infected when, as larvae, they were fed various concentrations of each virus, but significantly more larvae were removed from their cells when fed concentrated preparations of each virus than when fed diluted preparations. Susceptible larvae that became in apparently infected with KBV and SBV developed normally into in apparently infected pupae and later, emerged as in apparently infected worker bees.     

Detection of chronic bee paralysis virus and acute bee paralysis virus in Uruguayan honeybees

Chronic bee paralysis virus (CBPV) causes a disease characterized by trembling, flightless, and crawling bees, while Acute bee paralysis virus (ABPV) is commonly detected in apparently healthy colonies, usually associated to Varroa destructor. Both viruses had been detected in most regions of the world, except in South America. In this work, we detected CBPV and ABPV in samples of Uruguayan honeybees by RT-PCR. The detection of both viruses in different provinces and the fact that most of the analyzed samples were infected, suggest that, they are widely spread in the region. This is the first record of the presence of CBPV and ABPV in Uruguay and South America.     

Three-dimensional reconstruction of the Z disk of sectioned bee flight muscle

The three-dimensional structure of the central region of the Z disk of honeybee flight muscle has been determined to a resolution of 70 A by three-dimensional reconstruction from electron micrographs of tilted thin sections. The reconstructions show a complex assembly in which actin filaments terminate and are cross-linked together; a number of structural domains of this network are resolved in quantitative three-dimensional detail. The central region of the Z disk contains two sets of overlapping actin filaments of opposite polarity, which originate in the sarcomeres adjacent to the Z disk, and connections between these filaments. The filaments are deflected by the attachment of cross-links; spacing between filaments change by greater than 100 A during their passage through the Z disk. Each actin filament is linked by connecting structures to four filaments of opposite polarity and two filaments are of the same polarity. Four types of connecting density domain are observed in association with pairs of filaments of opposite polarity: C1, C2, C3, and C5. Two of these, C3 and C5, are associated with the ends of actin filaments. Another connection, C4, is associated with three filaments of the same polarity; C4 is threefold symmetric.     

Three-dimensional structure of physalis mottle virus: implications for the viral assembly.

The structure of the T=3 single stranded RNA tymovirus, physalis mottle virus (PhMV), has been determined to 3.8 A resolution. PhMV crystals belong to the rhombohedral space group R 3, with one icosahedral particle in the unit cell leading to 20-fold non-crystallographic redundancy. Polyalanine coordinates of the related turnip yellow mosaic virus (TYMV) with which PhMV coat protein shares 32 % amino acid sequence identity were used for obtaining the initial phases. Extensive phase refinement by real space molecular replacement density averaging resulted in an electron density map that revealed density for most of the side-chains and for the 17 residues ordered in PhMV, but not seen in TYMV, at the N terminus of the A subunits. The core secondary and tertiary structures of the subunits have a topology consistent with the capsid proteins of other T=3 plant viruses. The N-terminal arms of the A subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, have a conformation very different from the conformations observed in B and C subunits that constitute hexameric capsomers with near 6-fold symmetry at the icosahedral 3-fold axes. An analysis of the interfacial contacts between protein subunits indicates that the hexamers are held more strongly than pentamers and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. These observations suggest a plausible mechanism for the formation of empty capsids, which might be initiated by a change in the conformation of the N-terminal arm of the A subunits. The structure also provides insights into immunological and mutagenesis results. Comparison of PhMV with the sobemovirus, sesbania mosaic virus reveals striking similarities in the overall tertiary fold of the coat protein although the capsid morphologies of these two viruses are very different.     

Prevention of Chinese Sacbrood Virus Infection in Apis cerana using RNA Interference

Chinese sacbrood virus (CSBV) is the pathogen of Chinese sacbrood disease, which poses a serious threat to honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. Here we report on prevention of CSBV infection by feeding second instar larvae of A. cerana with specific sequences of CSBV double-stranded RNA (dsRNA). Protection of the bee larvae from CSBV by ingestion of CSBV-derived dsRNA was further demonstrated by quantitative real-time PCR (qRT-PCR) and northern blot analysis. The result provides a potential method to protect A. cerana from CSBV infection.     

Multiple virus infections in the honey bee and genome divergence of honey bee viruses

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

The incidence of virus diseases in the honey bee

Chronic bee-paralysis virus and sacbrood virus occur commonly in apparently normal honey-bee colonies in Britain. Most sick adult bees not affected by Nosema apis, Malpighamoeba mellificae or Acarapis woodi have chronic paralysis and most dead larvae not affected by micro-organisms have sacbrood. Both virus diseases are probably limited by hereditary factors, but unknown environmental factors also seem to influence disease. Paralysed bees from Australia, Canada, Eire, Germany and Mexico were found to be infected with chronic paralysis virus.     

Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.