Cydia Pomonella Granulosis Virus Research Articles

The major interest for crop protection of agrochemical substances without maximum residue limit (MRL)

Description of the subject. The real-time management of residues of plant protection products is a general issue for food producers and/or exporters. Authorized pesticides under European Regulation (EC) No 1107/2009 (Reg. 1107/2009) are granted a maximum residue limit (MRL) defined by the European Chemicals Agency (ECHA). These maximum residue limits are managed by the European Commission and listed in EC Regulation No 396/2005. MRLs for newly approved substances are set for pesticide residues present in products or groups of products covered by Annex I with a default value of 0.01 mg·kg-1, unless they have been considered, elaborated upon on approval and affected in one of the six other Annexes (II to VII). Substances listed in Annex IV are favoured in the selection of substances to be used by producers as it is for substances with very low potential impacts (low-risk substances [Art. 22 of Reg. 1107/2009] which therefore require no MRL; basic substances [Art. 23 of Reg. 1107/2009]; and microorganisms). The number of substances affected by this lack of an MRL (without residues survey requirement) has been steadily increasing since the publication of the SANCO/11188/2013 Guideline. Many biocontrol agents are listed in Annex IV, including microbial insecticides (e.g. Bacillus thuringiensis, Cydia pomonella granulosis virus), semiochemicals used in traps (e.g. ammonium acetate, putrescine) and some natural substances (e.g. sulphur) as well as all the currently listed basic substances. Objectives. To show that plant protection may be achieved with less concern or control (auto + external) than with certain agrochemical pesticides, including those without maximum residue limits (MRL). Method. Analysis of EU Pesticide Database and EU Regulation (EU) No 540/2011. Results. All approved pesticide active substances with no MRL included in Annex IV of Regulation (EC) No 396/2005, listed by substance status type (active, low-risk or basic) and category (microorganism, semiochemical or natural). Conclusions. These results may be used to help achieve plant protection with much less risk of contaminations, while noting the necessity for controls, analytical issues, positive controls, as well as the possibility of downgrading and withdrawal of approval from the market.

Successful Application of the Baculovirus Product Madex® for Control of Cydia Pomonella (L.) in Bulgaria

The codling moth (CM), Cydia pomonella (L.), causes heavy damage in Bulgarian apple orchards. Conventionally treated orchards, were monitored in this study. In spite of numerous chemical treatments, these orchards showed increasing flight densities of CM moths, growing populations of hibernating larvae and rising fruit damage rates. Thus, the control of CM by conventional spray- ing programmes became ineffective, apparently due to the development of resistance to insecticides. Products based on the Cydia pomonella granulosis virus (CpGV), such as Madex®, may provide alternative control tools that can be applied with other approaches, for a sustainable control strategy. The trials were carried out in Central-South and South-East Bulgaria, in 2006 -2010. Four treatments of Madex® against the first generation, and six treatments against the second generation kept the fruit damage and population density of CM at a low level. Based on the obtained results, different control strategies have been suggested, depending on the initial CM pressure in a particular orchard. Madex® may be a promising alternative to traditional programmes of CM control. Its dose, however, should be adjusted to the initial CM population density. Also, at a high or moderate CM population density Madex® applications should be combined with MD to avoid resistance of CM to granulovirus. At the peak of CM hatching, additional chemical treatments may be sometimes necessary. Such treatments include using insecticides which are still effective against CM.

Parallel acoustic detection of biological warfare agents surrogates by means of piezoelectric immunochips

This paper focuses on flow functionalization of piezoelectric immunochips with antibodies against four different biological warfare agents (BWA) surrogates. To perform parallel detection of all BWA surrogates at once, the E4 Quartz Crystal Microbalance with Dissipation monitoring system (QCM-D) is used. Assessment of antibodies immobilization, parallel detection of related BWA surrogates diluted in buffer solutions and regeneration of the complex antibodies/BWA surrogates are first discussed. Minimal detection thresholds for Escherichia coli MRE 162, Bacillus atrophaeus, Cydia pomonella granulosis virus (CpGV) and ovalbumin are respectively equal to 2.4 × 10 7 CFU/mL, 1.4 × 10 6 spores/mL, 1.1 × 10 8 granules/mL and 1 μg/mL. Detection experiments for three of the four BWA surrogates ( E. coli MRE 162, B. atrophaeus and ovalbumin) immersed in real liquid matrices from air sampler are successfully performed.

Complete sequence and transposon mutagenesis of the BamHI J fragment of Cydia pomonella granulosis virus.

The BamHI J fragment of Cydia pomonella granulosis virus was subcloned and subjected to transposon mutagenesis in Escherichia coli using a Tn3 derivative. After screening by restriction endonuclease digestion and polymerase chain reaction (PCR), 44 clones were selected representing insertions every 100 to 300 bp. The complete sequence was compiled and the transposon insertion sites mapped precisely by sequencing. Analysis of the sequence revealed the presence of 7 potential open reading frames (ORFs). The BamHI J fragment was already known to encode IAP and OPDV-E6. Three other ORFs encode products similar to known proteins, viz. an Autographa californica nuclear polyhedrosis virus 8.6 kDa protein, a Lymantria dispar nuclear polyhedrosis virus 34 kDa protein, and vertebrate reovirus omega-1 proteins. The ORF with similarity to omega-1 is also similar to baculovirus p10 proteins. In both cases, the similarity occurs in regions likely to form a coiled-coil structure.

TCl4.7: A Novel Lepidopteran Transposon Found in Cydia pomonella Granulosis Virus

After the co-infection of larvae of the lepidopteran Cryptophlebia leucotreta with the two baculoviruses C. leucotreta granulosis virus and Cydia pomonella granulosis virus (ClGV and CpGV, respectively), three CpGV mutants and one ClGV mutant carrying insertions of 0.9 to 4.7 kb have been isolated. By cloning, sequencing, and hybridization analysis, one of these insertions was identified as a transposon-like element derived from the C. leucotreta genome. This element, called TCl4.7, was found in the genome of CpGV which naturally replicates in C. pomonella. Sequence analysis suggested that TCl4.7 is 4726 bp in size, flanked by imperfect inverted terminal repeats of 29 bp, and integrated into the target dinucleotide TA. TCl47 encompasses an open reading frame sharing homologies to transposase genes of the Tc1-related transposable elements found in Caenorhabditis and in Drosophila species. The open reading frame might represent a pseudogene since it is missing an ATG start codon. The integration site of TCl4.7 is located in a non-protein-coding region of the CpGV genome at m.u. 9.5. In bioassays the TCl4.7-carrying virus and all the other mutants except for one showed LC 50 values similar to those of CpGV and ClGV. This is the first report of the horizontal escape of a transposable element during the in vivo infection of lepidopteran larvae by granulosis viruses.

The granulin gene region of Cryptophlebia leucotreta granulosis virus: sequence analysis and phylogenetic considerations

The nucleotide sequence of a 2.2 kb genome region of the baculovirus Cryptophlebia leucotreta granulosis virus (CIGV) encompassing the granulin gene and a second open reading frame, designated ORF909, was determined. The putative granulin ORF comprises 744 nucleotides encoding a polypeptide with a predicted M(r) of 29.3 K. The 5' leader of the granulin gene contains a canonical late baculovirus promoter (ATAAG) and differs only in two nucleotide positions from the sequence of Cydia pomonella granulosis virus (CpGV). Sequence comparison with the granulin genes of different granulosis viruses indicated a very close relationship between CIGV and CpGV of about 96% amino acid identity. ORF909 is 909 nucleotides in length and encodes a potential protein of M(r) 36.2K. It contains two zinc finger-like motifs, similar to ME53, which was previously described for Autographa californica multiple nucleocapsid nuclear polyhedrosis virus. ORF909 protein is significantly shorter than ME53 protein, lacking more than 105 amino acids of the amino terminus of ME53. Both genes contain an early and a late promoter motif suggesting a similar temporal regulation. Homologous sequences to ORF909 are also present upstream of the granulin genes of other granulosis viruses (GVs) indicating that this ORF is common in GVs.

An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs

Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA.

Replication of Cydia pomonella granulosis virus in cell cultures.

Several primary cell lines that support the complete replication of Cydia pomonella granulosis virus have been established from one culture of C. pomonella embryonic cells. Virus passaged three times in cells and once in larvae showed no change in restriction enzyme fragment patterns. Stages in virus replication observed by electron microscopy resembled those from in vivo studies. Cell lines that were maintained at or below 21 degrees C retained susceptibility to virus over a period of 4 years whereas the same cell lines maintained at 27 degrees C gradually lost their susceptibility and eventually could not be infected at all.

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.

Physical map of the Cryptophlebia leucotreta granulosis virus genome and its relationship to the genome of Cydia pomonella granulosis virus.

A physical map of the genome of Cryptophlebia leucotreta granulosis virus (CIGV) was constructed for the restriction enzymes BamHI, EcoRI, KpnI, NdeI, NruI, SacI and XhoI using hybridization techniques. The size of the viral genome was determined to be 112.4 kbp. A restriction fragment library covering almost the entire genome of CIGV was constructed, and the position of the granulin gene was identified by cross-hybridization with granulin coding fragments of Cydia pomonella granulosis virus (CpGV). Two further regions of intergenomic similarity between CIGV and CpGV were mapped. These regions were aligned and show a collinear arrangement.

Variation in Cydia pomonella Granulosis Virus Isolates and Physical Maps of the DNA from Three Variants

SUMMARY Cydia pomonella granulosis viruses (CpGV) from seven different sources in Europe, America and New Zealand were compared by restriction enzyme analysis. Most samples were indistinguishable from the Mexican isolate (CpGV-M). Isolates from Russia (CpGV-R) and England (CpGV-E) showed small genotypic differences. CpGV-E was shown to be a mixture of two variants, E1 and E2. CpGV-E1 was indistinguishable from CpGV-M. A physical map of CpGV-M was constructed for the enzymes EcoRI, BamHI, HindIII, SmaI and ApaI. A comparison of fragment profiles allowed construction of maps for CpGV-R and CpGV-E2. Relative to CpGV-M, CpGV-R had a single deletion of 2.4 kbp and CpGV-E2 was modified in one area resulting in an additional EcoRI site, a shift in a BamHI site and in total about 1 kbp more DNA. The map was orientated by locating the granulin gene using the cloned granulin gene from Trichoplusia ni GV as a probe. There was no significant difference between the infectivities of the Mexican, Russian and English isolates for neonate larvae.

In vitro replication of theCydia pomonella(codling moth) granulosis virus

Nuclear polyhedrosis virus (family Baculoviridae, subgroup A) in vitro systems have been well established for more than ten years, and have permitted application of modern microbiological techniques. Although widely tried, similar systems for granulosis virus (subgroup B) replication have not been obtained to our knowledge. We report here on the successful in vitro replication of Cydia pomonella granulosis virus in a Cydia pomonella primary cell line. The replication was confirmed by light and electron microscopy. This granulosis virus in vitro replication system will be an important tool for further granulosis virus research and for comparative studies on granulosis and nuclear polyhedrosis virus replication.