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.     

Prevalence of pathogenic bee viruses in Hungarian apiaries: situation before joining the European Union

A survey on the occurrence of six honeybee-pathogenic viruses was carried out using one-step RT-PCR assays. Samples were collected between 1999 and 2004 in 52 Hungarian apiaries located in different regions of the country. The results of the assays on samples of adult honeybees and Varroa destructor mites were compared to similar surveys from France and Austria. The study demonstrates geographical differences in the prevalence of honeybee viruses between Hungary and the older EU member states. The results could serve as a basis for monitoring further changes in the distribution of honeybee viruses in Europe.     

Formation of ethylene glycol and trimethylamine from choline by Candida tropicalis

Abstract The degradation of choline by Candida tropicalis cells grown in a medium containing choline as a nitrogen source was examined. The degradation of choline by resting cells was stimulated by the addition of Cu²⁺ or glutathione, and inhibited by 2-mercaptoethanol or potassium cyanide. With feeding of [1,2-¹⁴C]choline in the resting cell reaction, the release of ¹⁴C-labelled ethylene glycol was observed on radio-gas-liquid chromatography. Ethylene glycol, as one of the degradation products, was also observed on thin-layer and gas-liquid chromatographies, and mass spectrometry. Thus, it is suggested that choline is converted to ethylene glycol and trimethylamine by C. tropicalis .     

Plaque purification of cricket paralysis virus using an agar overlay on Drosophila cells

Cricket paralysis virus, an insect picorna-like virus, grown and assayed on Drosophila malanogaster cells, gave titers in excess of 10⁹ PFU/ml. The virus was plaque purified using an agar overlay and the intracellular polypeptides induced by “crude” and plaque-purified virions were demonstrated to be very similar.     

Honeybee viruses in Uruguay

Mortality of honeybees is a serious problem that beekeepers have to face periodically in Uruguay and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Chronic bee paralysis virus, Acute bee paralysis virus, Black queen cell virus, Sacbrood virus and Deformed wing virus in samples of Uruguayan honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in different provinces, simultaneous co-infection of colonies by several viruses and the fact that 96% of the samples were infected with one or more virus, indicates they are widely spread in the region.     

Improvement of RT-PCR detection of chronic bee paralysis virus (CBPV) required by the description of genomic variability in French CBPV isolates

A new RT-PCR test has been developed to diagnose Chronic bee paralysis virus (CBPV) that is able to detect genetically variable viral isolates. In fact, up to 8.7% divergence between partial nucleotide sequences from viral isolates from French honey bees was highlighted in a preliminary variability study. The previously-described RT-PCR was unable to detect all these viral isolates and RT-PCR diagnosis needed improvement. The new RT-PCR test can detect up to 40% more CBPV isolates.     

A survey for pathogens of the black field cricket, Teleogryllus commodus, in the Western District of Victoria,Australia

A pathogen survey of the black field cricket, Teleogryllus commodus, in the Western District of Victoria, Australia, during 1979 revealed that cricket paralysis virus (CrPV) was present in 42.7% of the 232 sites sampled. The fungus Metarhizium anisopliae was detected in 5.2% of the sites and represents a new pathogen record for T. commodus. The distribution of both pathogens throughout the sites sampled appeared to be random. There was a positive correlation between sample size and the likelihood of detecting a pathogen, while analysis showed that approximately 30% of the sites were probably virus free. The results are discussed in terms of the potential of CrPV and M. anisopliae as biological control agents for the black field cricket.     

Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites

Two exceptional mechanisms of eukaryotic translation initiation have recently been identified that differ fundamentally from the canonical factor-mediated, end-dependent mechanism of ribosomal attachment to mRNA. Instead, ribosomal 40S subunits bind in a factor-independent manner to the internal ribosomal entry site (IRES) in an mRNA. These two mechanisms are exemplified by initiation on the unrelated approximately 300 nt.-long Hepatitis C virus (HCV) IRES and the approximately 200 nt.-long cricket paralysis virus (CrPV) intergenic region (IGR) IRES, respectively. Ribosomal binding involves interaction with multiple non-contiguous sites on these IRESs, and therefore also differs from the factor-independent attachment of prokaryotic ribosomes to mRNA, which involves base-pairing to the linear Shine-Dalgarno sequence. The HCV IRES binds to the solvent side of the 40S subunit, docks a domain of the IRES into the ribosomal exit (E) site and places the initiation codon in the ribosomal peptidyl (P) site. Subsequent binding of eIF3 and the eIF2-GTP/initiator tRNA complex to form a 48S complex is followed by subunit joining to form an 80S ribosome. The CrPV IRES binds to ribosomes in a very different manner, by occupying the ribosomal E and P sites in the intersubunit cavity, thereby excluding initiator tRNA. Ribosomes enter the elongation stage of translation directly, without any involvement of initiator tRNA or initiation factors, following recruitment of aminoacyl-tRNA to the ribosomal aminoacyl (A) site and translocation of it to the P site.     

检测蜜蜂急性麻痹病毒TaqMan实时荧光RT-PCR方法的建立和初步应用

[背景] 蜜蜂急性麻痹病毒（Acute Bee Paralysis Virus,ABPV）是一种高毒力的蜜蜂病毒,可以引起蜜蜂的大批死亡和蜂群衰竭。[目的] 建立一种快速、灵敏的ABPV实时荧光RT-PCR检测方法。[方法] 根据ABPV衣壳蛋白基因保守序列设计引物和探针,通过对引物、探针浓度和退火温度等反应条件进行优化,建立基于TaqMan探针检测ABPV的实时荧光RT-PCR方法,并对方法的灵敏性、特异性和稳定性进行验证。[结果] ABPV实时荧光RT-PCR检测方法在9.8×10¹-9.8×10⁸ copies/μL之间呈现良好的线性关系,线性相关系数R²为0.998,扩增效率为103.8%。该方法的检测灵敏度为9.8 copies/μL;对其他蜜蜂病毒不发生交叉反应,具有良好的特异性;重复性试验结果显示组内和组间的变异系数分别为0.19%-0.80%和0.57%-1.07%,重复性良好。对2018年-2019年在福建地区采集的70份蜜蜂样品进行ABPV检测,阳性率为2.86%。[结论] 建立的ABPV实时荧光RT-PCR检测方法能用于该病的实验室检测、流行病学调查和疫情监测。     

Multiplex RT-PCR with broad-range primers and an exogenous internal amplification control for the detection of honeybee viruses in bumblebees

Bumblebees are commercially reared and transported worldwide mainly for pollination of greenhouse tomatoes. Three honeybee viruses have been reported in bumblebees: Acute bee paralysis virus, Kashmir bee virus and Deformed wing virus. We developed a multiplex RT-PCR with primers designed on highly conserved regions of the RNA-dependent RNA polymerase in order to detect a maximum range of viral variants. Rearing facilities and governmental organizations can now thoroughly screen bumblebee colonies with a cost-effective technique with an integrated internal amplification control (IAC) implementable in laboratories that strive for International Organization for Standardization (ISO) certification.     

Survey of six bee viruses using RT-PCR in Northern Thailand

Six honey bee viruses were surveyed using RT-PCR in Northern Thailand where about 80% of Thai apiaries are located. Tested samples were found to be positive for deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and Kashmir bee virus (KBV). In the collected samples, neither chronic bee paralysis virus nor black queen cell virus nucleic acids could be detected. It was found that DWV was the most widespread and ABPV was the second most prevalent. Kashmir bee virus was found only in the Lampang province where high infestation of Varroa destructor mite occurred. Tropilaelaps, European foulbrood, and Chalkbrood diseases were found in some apiaries.     

Characterisation and serological relationships of strains of Kashmir bee virus

Isolates of novel strains of Kashmir bee virus (KBV) were obtained from field-collected dead adults of Apis mellifera from honey bee colonies in Canada and Spain. They differed from other strains of KBV in their tendency to aggregate in dilute buffer solution and in containing only three proteins when analysed by SDS-polyacrylamide gel electrophoresis compared with five proteins resolved in the type strain of KBV from Apis cerana in India and six proteins in KBV strains from South Australia and New Zealand. Immunodiffusion tests and Western blotting studies indicated that the five virus isolates were serologically related and all were related to acute paralysis virus (APV). The world distribution of KBV strains and their apparent relationship with APV are discussed.     

Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus

The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA.     

北京地区六种蜜蜂病毒病的流行病学研究

【目的】对蜜蜂的6种病毒:以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)、残翅病毒(Deformed wing virus,DWV)、囊状幼虫病病毒(Sacbrood virus,SBV)、急性蜜蜂麻痹病毒(Acute bee paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、慢性麻痹病毒(Chronic bee paralysis virus,CBPV)在北京地区的流行情况进行调查,以期为该地区蜜蜂病毒病的防控提供一定的理论依据。【方法】应用多重RT-PCR法确定上述6种病毒在该地区的感染情况,并通过序列分析确定特异性。【结果】在所有检测样本中均未检测到急性麻痹病病毒和慢性麻痹病病毒,感染率最高的是以色列急性麻痹病毒,其次是残翅病毒。检测的样本普遍存在混合感染。【结论】以色列急性麻痹病毒、残翅病毒、囊状幼虫病病毒、黑蜂王台病毒4种病毒可能在北京地区广泛分布。     

凝血、纤溶和纤维化与慢性乙型肝炎病毒感染状态的关系

目的:探究凝血、纤溶和纤维化与慢性乙型肝炎病毒感染状态的关系。方法:选取我院收治的慢性乙型病毒肝炎患者120例,经检测后将患者分为免疫清除组40例、免疫耐受组40例、非活动性组40例和对照组40例,分别检测每组患者的凝血功能、纤溶和纤维化的水平以及统计与感染状态的相关程度。结果:与对照组相比,其余各组凝血功能降低(P0.05),纤溶和纤维化水平明显升高(P0.05);与非活动组和免疫耐受组相比,免疫清除组患者的各项指标变化明显(P0.05);非活动组与免疫耐受组相比各项指标大多无统计学差异(P0.05);经统计凝血、纤溶和纤维化与HBV具有相关性,凝血功能与HBV呈负相关,纤溶与HBV呈正相关,纤维化与HBV呈正相关。结论:患者慢性乙型肝炎病毒感染状态不同,其凝血、纤溶和纤维化水平含量有明显差异,可作为疾病发展的监测依据,临床上值得推广。     

Increased serum oxysterol concentrations in patients with chronic hepatitis C virus infection

Oxidative stress and dysregulated cholesterol metabolism are characteristic features of chronic hepatitis C virus infection (CHC). Therefore, we analyzed serum oxysterol profiles in CHC patients and examined the significance of oxysterols in CHC. The concentrations of 7α-hydroxycholesterol, 4β-hydroxycholesterol and 25-hydroxycholesterol as determined by LC–ESI–MS/MS were significantly elevated by +236%, +29% and +44%, respectively, in CHC patients compared with controls. Moreover, the elevated levels were significantly decreased by anti-viral therapy using PEGylated-interferon and ribavirin for 3 months. In contrast, 24S-hydroxycholesterol, 27-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one concentrations were not affected by CHC or anti-viral treatment. These results suggest that some oxysterols that are elevated in CHC are produced by cholesterol autoxidation due to oxidative stress or inflammation in the liver. Oxysterols may represent novel targets for the inhibition of disease progression and the prevention of hepatocarcinogenesis in CHC patients.     

Recent advances in research on hepadnaviral infection in the woodchuck model

The woodchuck model is an excellent animal model to study hepadnaviral infection. The new progresses in this model made possible to examine the T-cell mediated immune responses in acute and chronic hepadnaviral infection. Recently, a new assay for cytotoxic T-cells based on detection of CD107 was established for the woodchuck model. In addition, new immunotherapeutic approaches based on combination of potent antiviral treatment and DNA-protein vaccines were proven to be useful for treatment of chronic hepatitis B.