Prevalence and phylogeny of Kakugo virus, a novel insect picorna-like virus that infects the honeybee (Apis mellifera L.), under various colony conditions

We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), from the brains of aggressive worker honeybees that had counterattacked a giant hornet. To survey the prevalence of KV in worker populations engaged in various labors, we quantified KV genomic RNA. KV was detected specifically from aggressive workers in some colonies, while it was also detected from other worker populations in other colonies where the amount of KV detected in the workers was relatively high, suggesting that KV can infect various worker populations in the honeybee colonies. To investigate whether the KV strains detected were identical, phylogenetic analysis was performed. There was less than a 2% difference in the RNA-dependent RNA polymerase (RdRp) sequences between KV strains from aggressive workers and those from other worker populations, suggesting that all of the viruses detected were virtually the same KV. We also found that some of the KV-infected colonies were parasitized by Varroa mites, and the sequences of the KV strains detected from the mites were the same as those detected from the workers of the same colonies, suggesting that the mites mediate KV prevalence in the honeybee colonies. KV strains had approximately 6% and 15% sequence differences in the RdRp region from deformed wing virus and Varroa destructor virus 1, respectively, suggesting that KV represents a viral strain closely related to, but distinct from, these two viruses.     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.     

Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans.

The processing of the Sindbis virus nonstructural polyprotein translated in vitro has been studied. When Sindbis virus genomic RNA was translated in a reticulocyte lysate, polyprotein P123 was cleaved efficiently to produce nsP1, nsP2, and nsP3. Inhibition of this processing by anti-nsP2 antibodies, but not by antibodies specific for nsP1, nsP3, or nsP4, suggested that the viral proteinase was present in nsP2. To localize the proteolytic activity more precisely, deletions were made in a full-length cDNA clone of Sindbis virus, and RNA was transcribed from these constructs with SP6 RNA polymerase and translated in vitro. Although virtually all of the nsP1, nsP3, and nsP4 sequences could be deleted without affecting processing, deletions in the N-terminal half of nsP2 led to aberrant processing, and deletions in the C-terminal half abolished proteolysis. However, inactive polyproteins containing the nsP2 deletions could be processed by exogenously supplied proteins translated from virion RNA, demonstrating that cleavage was virus specific and not due to a protease present in the reticulocyte lysate and that the deleted polyproteins still served as substrates for the enzyme. From these results and from experiments in which processing was studied at increasingly higher dilution, we have concluded the following: (i) the viral nonstructural proteinase is located in the C-terminal half of nsP2; (ii) in the P123 precursor the cleavage between nsP2 and nsP3 occurs efficiently as a bimolecular reaction (in trans) to remove nsP3, while the bond between nsP1 and nsP2 is cleaved inefficiently, but detectably, in trans, but no autoproteolysis of P123 was detected; (iii) once nsP3 has been removed, the bond between nsP1 and nsP2 in the P12 precursor is cleaved efficiently by autoproteolysis (in cis). This mode of processing leads to a slow rate of cleavage, particularly early in infection, suggesting that the polyproteins might play roles in virus RNA replication distinct from those of the cleaved products. A hypothesis is presented that the proteinase is a thiol protease related to papain.     

The effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees (Apis mellifera L.)

Elucidating the mechanisms by which honey bees process pollen vs. protein supplements are important in the generation of artificial diets needed to sustain managed honeybees. We measured the effects of diet on protein concentration, hypopharyngeal gland development and virus titers in worker honey bees fed either pollen, a protein supplement (MegaBee), or a protein-free diet of sugar syrup. Workers consumed more pollen than protein supplement, but protein amounts and size of hypopharyngeal gland acini did not differ between the two feeding treatments. Bees fed sugar syrup alone had lower protein concentrations and smaller hypopharyngeal glands compared with the other feeding treatments especially as the bees aged. Deformed wing virus was detected in workers at the start of a trial. The virus concentrations increased as bees aged and were highest in those fed sugar syrup and lowest in bees fed pollen. Overall results suggest a connection between diet, protein levels and immune response and indicate that colony losses might be reduced by alleviating protein stress through supplemental feeding.     

RNAi沉默王浆主蛋白1(Mrjp1)基因对意大利蜜蜂工蜂学习和记忆的影响

【目的】前期研究发现经吡虫啉处理的意大利蜜蜂Apis mellifera ligustica(简称“意蜂”)工蜂学习能力下降,转录组学分析表明王浆主蛋白1(major royal jelly protein 1, MRJP1)基因在吡虫啉处理的蜜蜂脑中显著下调,MRJP1可能参与调控蜜蜂学习能力。本研究旨在采用RNA干扰(RNAinference, RNAi)技术将Mrjp1特异性沉默,验证MRJP1在意蜂工蜂嗅觉学习中的关键作用。【方法】通过克隆技术获得Mrjp1基因cDNA 序列,经测序验证后,设计引物,合成用于RNAi干扰Mrjp1基因表达的dsRNA。注射dsMrjp1的意蜂工蜂作为处理组(dsMrjp1注射组),注射dsEGFP的意蜂工蜂作为对照组(dsEGFP注射组),随后通过伸吻反应(proboscis extension response, PER)实验比较两组的嗅觉学习与记忆能力差异。最后采用实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测注射dsMrjp1后意大利蜜蜂工蜂脑中Mrjp1的相对表达量。【结果】dsMrjp1注射组与dsEGFP注射组意蜂工蜂学习能力差异显著,dsMrjp1注射组意蜂工蜂的学习能力显著降低。学习后2 h,两组意蜂工蜂的记忆力无显著差异。qRT-PCR结果显示Mrjp1的表达水平在dsMrjp1注射组意蜂工蜂脑中显著低于dsEGFP注射组,表明学习能力降低的处理组意蜂脑内对应的Mrjp1表达水平也降低。【结论】通过RNAi抑制意蜂工蜂Mrjp1基因的表达后,其嗅觉学习能力受到显著性抑制,但记忆力未受到显著影响,提示Mrjp1可能是调控意蜂学习的重要基因之一。本研究结果有助于后续进一步研究蜜蜂嗅觉学习相关的分子机制。     

Identification and validation of reference genes for real time PCR expression studies in heads of nurse honeybees

The primary aim of this study was to identify reference genes and workers of particular role and ages that would be suitable for exploring genetic/epigenetic variations in constitutive expression of a gene encoding antimicrobial peptide defensin1 in worker heads using real-time PCR. This peptide is an integral component of larval food and honey and has potential to act against some brood pathogens. Expression levels of distinct genes may vary in worker heads due to genetic factors, age of bee, and particular role of a worker that depends on its age or colony needs. Prerequisite for exploring the variations in defensin1 expression was therefore to identify such workers in which correlated expression of defensin1 and suitable reference genes occurs. Selection process was done by carefully designed quantitative real-time PCR procedure in two colonies showing different age-related division of labor. Expression of ten candidate reference genes, defensin1 and amylase, as a marker of forager bees, was assessed in pooled head samples of workers aged 2 to 30 days. Correlated and moreover stable expression of defensin1 and six candidate genes was detected in nursing bees in both colonies. The suitable reference genes were therefore selected on the basis of their expression stability. This was evaluated by geNorm and NormFinder algorithms in pooled head samples and through plotted Cq data in head samples of individual nurse bees. As the best reference genes were selected: psa1, tctp1, cyclophilin, gapdh and mrjp4 (in this order). They are suitable for aforementioned defensin1 expression studies and also for studies of other genes expressed in heads of nurses. In addition, an amylase expression-based procedure for reliable distinguishing nurses from foragers was elaborated.     

Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.     

High-performance liquid chromatography (HLPC) measurement of catecholamines in single honeybee brains reveals caste-specific differences between worker bees and queens in apis mellifera

1. Catecholamines were determined by HPLC in honeybee brains using automatized extraction and on-line detection with an electrochemical detector.2. This method has high sensitivity in the range of fmols. Thus, catechols can be measured in single brains as well as in parts of brains.3. The comparison of brains of worker bees and queens shows a caste-specific content of eatecholamine concentrations in worker bees and queens.4. The amount of norepinephrine and dopamine are higher in queens than in worker bees.5. The differences are not caused by the different size of the respective brains, but reflect a difference in the two female castes.     

Effects of sacbrood virus on adult honey-bees

Much sacbrood virus accumulates in the brains of infected bees, especially of drones, without causing symptoms. However, infected individuals fly earlier in life than healthy bees and most infected foragers fail to collect pollen, as do bees briefly anaesthetized with CO₂. The few infected bees that gather pollen contaminate their loads with much sacbrood virus. Infection does not shorten the lives of drones, or of workers that have been deprived of pollen, but it much shortens the prolonged lives of workers that have eaten pollen. Infected workers, healthy workers deprived of pollen, and senile individuals are unable to maintain the usual metabolic rates of bees at temperatures below 35^oC, or to resist chilling.     

Synthesis and Cleavage of Influenza Virus Proteins

The NWS strain of influenza virus grows rapidly in and kills the MDCK dog kidney cell strain. Within 1 to 2 hr, the virus inhibits host cell protein synthesis and for 3 to 4 hr more it directs the synthesis of influenza virus proteins at a rate about twice that of uninfected cell synthesis. The rates of virus ribonucleic acid (RNA) and protein synthesis reach a maximum within the first few hours after infection and then drop. Plaque assays exhibit a linear dose-response, indicating that only one virion is necessary for productive infection. We have confirmed earlier reports regarding the fragmented nature of the RNA genome of purified influenza virions. However, high resolution gel electrophoresis indicated that each size class of viral RNA is heterogenous, so that there are at least 10 and probably more fragment sizes of RNA in these virions. Repeated attempts to detect infectivity in preparations of extracted viral RNA were completely negative (over a 10(8)-fold loss of infectivity after extraction). Even infection of the "infectious" RNA-treated cells with intact, related, influenza viruses failed to support infectivity of the isolated RNA or to rescue a host range genetic marker of the RNA. Purified influenza virions exhibit only three major protein peaks based on separation according to molecular weights. These three major virion proteins are the only major virion proteins synthesized in infected cells. This is true throughout the infectious cycle from several hours after infection until the cells are dying. However, the molecular weight of these virion proteins differs slightly depending upon the cell type in which the virus is grown. No host membrane proteins are incorporated into the virions as they bud through the cell membrane. Pulse-chase labeling early after infection or prolonged chase experiments indicate that influenza virus proteins are cleaved from one or more precursor polypeptides. In fact, each of the three major peaks seems to be a heterogeneous mixture of polypeptides in various stages of cleavage. Peptide analysis confirms that the three major peaks share common peptides, but the exact precursor product relationships are not clear. There may be one or several precursor proteins. Also there could be overlapping messenger RNA molecules of varying length giving rise to polypeptides of various sizes and overlapping sequences. Late in infection, amino acid labeling shows a preponderance of internal nucleocapsid protein synthesis, indicating that either this protein is much more stable to cleavage in infection or it is made from a more stable messenger. There is no obvious relationship between virion RNA fragments and viral protein sizes, so these fragments may be artifacts.     

Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.     

Ribonucleic Acid and protein synthesis in chick embryo cells infected with fowl plague virus

Fowl plague virus comprised four major protein components and several minor ones, two strains of the virus giving similar results. One of the components was identified as the nucleocapsid protein. Synthesis of the virion proteins could readily be detected in infected cells 3 hr after infection. The two subcellular fractions associated with viral ribonucleic acid (RNA) polymerase activity (nuclei and ribosomal pellet) were associated with the protein of the nucleocapsid and a second virion protein of unidentified function. Measurement of viral RNA and protein synthesis in cells infected with preparations of ultraviolet irradiated virus showed that the capacity to synthesise the RNA and protein species of highest molecular weight was lost most quickly, suggesting that the pieces of viral RNA function independently.     

Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly

The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.     

Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus: association of VP1 protein with RNA polymerase activity.

The bluetongue virus core particles have been shown to contain an RNA-directed RNA polymerase (1). To identify the protein responsible for the virion RNA polymerase activity, the complete 3.9 Kb DNA clone representing the largest RNA segment 1 (L1) of bluetongue virus (BTV-10) was placed under control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The derived recombinant virus was used to infect Spodoptera frugiperda cells. As demonstrated by stained polyacrylamide gel electrophoresis and by the use of bluetongue virus antibody, infected insect cells synthesized the largest protein of BTV-10 (VP1, 150 k Da). Antibody raised in rabbit to recombinant VP1 protein recognized bluetongue virus VP1 protein. The recombinant virus infected cell lysate had significantly inducible levels of RNA polymerase enzymatic activity as determined by a poly (U)-oligo (A) polymerase assay. The availability of enzymatically active bluetongue virus RNA polymerase provides a system in which we can precisely delineate the role this protein plays in the regulation of bluetongue replication.