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Transmission of viruses by Varroa destructor Anderson & Trueman to bees has been identified as one of the main threats to Apis mellifera L. colonies. It has also been suggested that synergistic parasite-pathogen interaction mediated by A. mellifera immunity can drive the collapse of honey bee colonies. We analyzed the relationship between deformed wing virus (DWV) viral load and gene expression levels of Toll Wheeler, cactus, domeless, TEPA, nitric oxide synthase, hymenoptaecin, vitellogenin, defensin, abaecin in winter bee samples of A. mellifera with and without deformed wings. Samples were collected from hives in the field with high V. destructor infestation. In bees with deformed wings we found an increase in DWV titre and upregulation of genes in the Jak-STAT signalling pathway. [ABSTRACT FROM AUTHOR]

Israeli acute paralysis virus (IAPV) can cause a systemic infection, resulting in mortality in both Apis and Bombus spp. bees. However, little is known about the virus infection dynamics within bee tissues. Here, we established systemic IAPV infections in reared bumblebee Bombus terrestris workers through feeding and injection and investigated the mortality, tissue tropism and viral localization. Injection of approximately 500 IAPV (IAPV inj stock) particles resulted in acute infection, viral loads within tissues that were relatively stable from bee to bee, and a distinctive tissue tropism, making this method suitable for studying systemic IAPV infection in bumblebees. Feeding with approximately 1 × 10 6 particles of the same virus stock did not result in systemic infection. A high-concentration stock of IAPV (IAPV fed stock) allowed us to feed bumblebees with approximately 1 × 10 9 viral particles, which induced both chronic and acute infection. We also observed a higher variability in viral titers within tissues and less clear tissue tropism during systemic infection, making feeding with IAPV fed stock less optimal for studying IAPV systemic infection. Strikingly, both infection methods and stocks with different viral loads gave a similar viral localization pattern in the brain and midgut of bumblebees with an acute infection. The implications of these findings in the study of the local immunity in bees and barriers to oral transmission are discussed. Our data provide useful information on the establishment of a systemic viral infection in bees. [ABSTRACT FROM AUTHOR]

Eleven viral isolates derived mostly in albopictus C6/36 cells from mosquito pools collected in Southeast Asia and the Americas between 1966 and 2014 contained particles with electron microscopy morphology typical of reoviruses. Metagenomics analysis yielded the near complete genomes of three novel reoviruses, Big Cypress orbivirus, Ninarumi virus, and High Island virus and a new tetravirus, Sarawak virus. Strains of previously characterized Sathuvarachi, Yunnan, Banna and Parry’s Lagoon viruses ( Reoviridae ), Bontang virus ( Mesoniviridae ), and Culex theileri flavivirus ( Flaviviridae) were also characterized. The availability of these mosquito virus genomes will facilitate their detection by metagenomics or PCR to better determine their geographic range, extent of host tropism, and possible association with arthropod or vertebrate disease. [ABSTRACT FROM AUTHOR]

The Chinese sacbrood virus (CSBV) was first isolated from Apis cerana in 1972. However, the biological characteristics of the CSBV that naturally infects Apis mellifera , causing larval death, have not been reported yet. In the present study, natural CSBV infection was evaluated using clinical symptoms of A. mellifera larvae, RT-PCR, electron microscopy, agar gel immunodiffusion assays, and virus analysis in inoculated A. cerana larvae. The isolated CSBV strain was named AmCSBV-SDLY-2016. Subsequently, AmCSBV-SDLY-2016 was analyzed by constructing a phylogenetic tree using VP1. Data from the phylogenetic tree suggested that AmCSBV-SDLY-2016 is evolutionarily close to JLCBS-2014. It was also observed that CSBV crossed the species barrier, causing the death of A. mellifera larvae. [ABSTRACT FROM AUTHOR]

The nucleotide and amino acid sequence of the ecdysteroid UDP-glucosyltransferase (egt), polyhedrin (pol), inhibitor of apoptosis (iap2 and iap3), late expression factor (lef) 9 and vp1054 genes of a SouthAfrican isolate of Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) was compared to a Canadian isolate ofTnSNPV. Nucleotide and amino acid sequence identity for the compared genes ranged from 89%to 98%and 95%to 99%, respectively. The deduced amino acid sequences of the TnSNPV egt, pol, iap3, lef-9 and vp1054 genes were used to infer phylogenetic trees and these were compared to the tree inferred from combined data sets consisting of the amino acid sequences of polyhedrin/granulin, lef-8 and lef-9 genes of 48 completely sequenced baculoviruses. The topologies of trees for the baculovirus core genes pol, lef-9 and vp1054 were better resolved than that of the auxiliary genes iap2, iap3 and egt when compared to the tree inferred from complete genome sequences. Sequence and phylogenetic analysis confirms that the two geographically disparate isolates are closely related. The tree inferred from the combined data set represents a quick and reliable method of identification particularly, when whole genome data are not available. [ABSTRACT FROM AUTHOR]

Within any one habitat, the relative fitness of organisms in a population can vary substantially. Social insects like the common wasp are among the most successful invasive animals, but show enormous variation in nest size and other fitness-related traits. Some of this variation may be caused by pathogens such as viruses that can have serious consequences in social insects, which range from reduced productivity to colony death. Both individual immune responses and colony-level traits such as genetic diversity are likely to influence effects of pathogen infections on colony fitness. Here we investigate how infections with Kashmir Bee Virus (KBV), immune response and intracolony genetic diversity (due to queen polyandry) affect nest size in the invasive common wasp Vespula vulgaris. We show that KBV is highly prevalent in wasps and expression of antiviral immune genes is significantly increased with higher viral loads across individuals. Patriline membership within a nest did not influence KBV susceptibility or immune response. A permutational MANCOVA revealed that polyandry, viral load and expression of the immune gene Dicer were significant predictors of variation in nest size. High intracolony genetic diversity due to polyandry has previously been hypothesized to improve colony-level resistance to parasites and pathogens. Consistent with this hypothesis, we observed genetically diverse colonies to be significantly larger and to produce more queens, although this effect was not driven by the pathogen we investigated. Invasive wasps clearly suffer from pathogens and expend resources, as indicated here by elevated immune gene expression, toward reducing pathogen-impact on colony fitness. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5 kb (viral DNA 1, VD1) and 6.0 kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22–24 nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism. [ABSTRACT FROM AUTHOR]

Viruses are one of the major threats for honeybees and until now more than 20 different viruses have been discovered. Viruses and their hosts are engaged in a continuous arms race in which viral defense mechanisms drive the adaptive evolution of host immune genes, which in turn results in counter-adaptations of the viral immune antagonists. The honeybee immune responses to non-viral pathogens have been extensively studied, but little is known about the antiviral responses. Recent evidence suggests that the main mechanism of antiviral defense in insects is the RNA interference (RNAi) pathway. Furthermore, there is evidence that some viruses suppress this RNAi pathway in order to evade antiviral immunity. In the present study, we test this hypothesis by comparing the gene expression levels of some key components of the RNAi response of honeybees that were naturally infected with at least five viruses with those who were infected with only two or three viruses using a colorimetric microarray developed in-house, called BeeClinic, and subsequently confirmed by quantitative reverse-transcription-polymerase chain reaction (qRT-PCR). Our results show that key components of the RNAi pathway are indeed downregulated in highly infected bees. We were able to show that high virus loads suppress key RNAi components, which results in a counteraction of the host RNAi antiviral defense. As the RNAi is a primary defense against viruses, these findings shed new light on pathogen-host interactions and can help mitigate escalating colony losses worldwide. [ABSTRACT FROM AUTHOR]

The Antheraea pernyi nucleopolyhedrovirus (ApNPV) is an exclusive pathogen of A. pernyi. The intense interactions between ApNPV and A. pernyi cause a series of physiological and pathological changes to A. pernyi. However, no detailed report exists regarding the molecular mechanisms underlying the interactions between ApNPV and A. pernyi. In this study, four cDNA libraries of the A. pernyi midgut, including two ApNPV-infected groups and two control groups, were constructed for transcriptomic analysis to provide new clues regarding the molecular mechanisms that underlie these interactions. The transcriptome of the A. pernyi midgut was de novo assembled using the Trinity platform because of the lack of a genome resource for A. pernyi. Compared with the controls, a total of 5,172 differentially expressed genes (DEGs) were identified, including 2,183 up-regulated and 2,989 down-regulated candidates, of which 2,965 and 911 DEGs were classified into different GO categories and KEGG pathways, respectively. The DEGs involved in A. pernyi innate immunity were classified into several categories, including heat-shock proteins, apoptosis-related proteins, serpins, serine proteases and cytochrome P450s. Our results suggested that these genes were related to the immune response of the A. pernyi midgut to ApNPV infection via their essential roles in regulating a variety of physiological processes. Our results may serve as a basis for future research not only on the molecular mechanisms of ApNPV invasion but also on the anti-ApNPV mechanism of A. pernyi. [ABSTRACT FROM AUTHOR]

Tetraviruses are small, non-enveloped, RNA viruses that exclusively infect lepidopteran insects. Their particles comprise 240 copies of a single capsid protein precursor (CP), which undergoes autoproteolytic cleavage during maturation. The molecular mechanisms of capsid assembly and maturation are well understood, but little is known about the viral infectious lifecycle due to a lack of tissue culture cell lines that are susceptible to tetravirus infection. We show here that binding and entry of the alphatetravirus, Helicoverpa armigera stunt virus (HaSV), is triggered by alkaline pH. At pH 9.0, wild-type HaSV virus particles undergo conformational changes that induce membrane-lytic activity and binding to Spodoptera frugiperda Sf9 cells. Binding is followed by entry and infection, with virus replication complexes detected by immunofluorescence microscopy within 2 h post-infection and the CP after 12 h. HaSV particles produced in S. frugiperda Sf9 cells are infectious. Helicoverpa armigera larval virus biofeed assays showed that pre-treatment with the V-ATPase inhibitor, Bafilomycin A1, resulted in a 50% decrease in larval mortality and stunting, while incubation of virus particles at pH 9.0 prior to infection restored infectivity. Together, these data show that HaSV, and likely other tetraviruses, requires the alkaline environment of the lepidopteran larval midgut for binding and entry into host cells. [ABSTRACT FROM AUTHOR]

Background: Culex Flavivirus (CxFV) is an insect-specific virus that is widely distributed and primarily infects mosquito species from the genus Culex. Its hosts include Culex tritaeniorhynchus, Culex quinquefasciatus, and Anopheles sinensis mosquitoes. Since its original identification, CxFV has been reported in several countries. Despite the increasing number of reports on CxFV, little is known about its genomic characteristics. It is unclear whether the phylogenetic relationships between the strains are influenced by host species and geographic location. Results: We characterized the Brazilian CxFV strain and performed a comprehensive genetic and phylogenetic characterization of CxFV based on all ORF sequences described so far. Our results revealed that the Brazilian strain is in a monophyletic clade with the Mexican strain. Overall, selective pressure indicates that the ORF is undergoing purifying selection. Conclusions: The phylogenetic analysis revealed a strong association between climate and CxFV ancestry. Also, based on phylogeny and the genetic distance between the main branches of the tree, we propose the classification of the available sequences into two different genotypes. We also suggest the existence of two different subtypes within Genotype 1. [ABSTRACT FROM AUTHOR]

Viral diseases are one of the multiple factors associated with honeybee colony losses. Apart from their innate immune system, including the RNAi machinery, honeybees can use secondary plant metabolites to reduce or fully cure pathogen infections. Here, we tested the antiviral potential of Laurus nobilis leaf ethanolic extracts on forager honeybees naturally infected with BQCV (Black queen cell virus). Total viral loads were reduced even at the lowest concentration tested (1 mg/ml). Higher extract concentrations (≥5 mg/ml) significantly reduced virus replication. Measuring vitellogenin gene expression as an indicator for transcript homeostasis revealed constant RNA levels before and after treatment, suggesting that its expression was not impacted by the L. nobilis treatment. In conclusion, plant secondary metabolites can reduce virus loads and virus replication in naturally infected honeybees. [ABSTRACT FROM AUTHOR]

Insects represent over 70% of all animal species. Recent virome analyses reveal unprecedented genetic diversity of insect viruses, which appears to match that of their hosts. Thus, insect-virus interactions may provide information on a vast repertoire of antiviral immune mechanisms. Tapping into this diversity is challenging because of several constraints imposed by the uniqueness of each insect model. Nevertheless, it is clear that many conserved and divergent pathways participate in the control of viral infection in insects. Co-evolution between hosts and viruses favors the development of immune evasion mechanisms by the pathogen. Viral suppressors can offer unique perspective on host pathways and emphasize the importance of RNA interference, apoptosis, but also NF-κB pathways and translation control in insect antiviral immunity. [ABSTRACT FROM AUTHOR]

A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n=42, year 2: n=132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n=20, year 2: n=15) tested negative for presence of X. fastidiosa. Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves. [ABSTRACT FROM AUTHOR]

Since its discovery, RNA interference has been identified as involved in many different cellular processes, and as a natural antiviral response in plants, nematodes, and insects. In insects, the small interfering RNA (siRNA) pathway is the major antiviral response. In recent years, the Piwi-interacting RNA (piRNA) pathway also has been implicated in antiviral defense in mosquitoes infected with arboviruses. Using Drosophila melanogaster and an array of viruses that infect the fruit fly acutely or persistently or are vertically transmitted through the germ line, we investigated in detail the extent to which the piRNA pathway contributes to antiviral defense in adult flies. Following virus infection, the survival and viral titers of Piwi, Aubergine, Argonaute-3, and Zucchini mutant flies were similar to those of wild type flies. Using next-generation sequencing of small RNAs from wild type and siRNA mutant flies, we showed that no viral-derived piRNAs were produced in fruit flies during different types of viral infection. Our study provides the first evidence, to our knowledge, that the piRNA pathway does not play a major role in antiviral defense in adult Drosophila and demonstrates that viralderived piRNA production depends on the biology of the host-virus combination rather than being part of a general antiviral process in insects. [ABSTRACT FROM AUTHOR]

In El Bajo Papaloapan, the main producing area of pineapple of Mexico, leaves with typical symptoms of viral infection consisting in chlorosis, flaccidity, reduced growth and reddening were collected. By RT-PCR with specific primers for the hsp70 gene and subsequent sequencing were detected Pineapple mealybug wilt virus associated-virus 1 (PMWaV-1) and Pineapple mealybug wilt virus associated-3 (PMWaV-3). From the sequences obtained a tree was done with sequences from different regions of the world available in GenBank in order to know their similarity. The sequence obtained from the Mexican isolate PMWaV-1 was genetically related to the sequences of isolates from Cuba, Taiwan, Thailand and Hawaii and more distant from the Australian isolate. The sequence obtained for the Mexican isolate PMWaV-3 was more related to isolates from Hawaii, Cuba, Australia and Taiwan and more distant from the Thailand isolate. This is the first report of the presence of these two viruses in Mexico. [ABSTRACT FROM AUTHOR]

Historically an ectoparasite of the native Giant honey bee Apis dorsata , the mite Tropilaelaps mercedesae has switched hosts to the introduced western honey bee Apis mellifera throughout much of Asia. Few data regarding lethal and sub-lethal effects of T. mercedesae on A. mellifera exist, despite its similarity to the devastating mite Varroa destructor . Here we artificially infested worker brood of A. mellifera with T. mercedesae to investigate lethal (longevity) and sub-lethal (emergence weight, Deformed wing virus (DWV) levels and clinical symptoms of DWV) effects of the mite on its new host. The data show that T. mercedesae infestation significantly reduced host longevity and emergence weight, and promoted both DWV levels and associated clinical symptoms. Our results suggest that T. mercedesae is a potentially important parasite to the economically important A. mellifera honey bee. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus (BmBDV), which causes fatal flacherie disease in the silkworm, replicates only in midgut columnar cells. The viral resistance expressed by some silkworm strains, which is characterized as non-susceptibility irrespective of the viral dose, is determined by a single gene, nsd - 2 . We previously identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter that might function as a receptor for BmBDV. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 (resistance gene), + nsd-2 (susceptibility gene) and BmBDV propagation. Quantitative RT-PCR (qRT-PCR) analysis using total RNA isolated from the anterior, middle, and posterior parts of the midgut showed that nsd-2 and + nsd-2 were strongly expressed in the posterior part of the midgut. The expression levels of both genes were very low in the anterior and middle parts. The qRT-PCR analysis showed that the expression levels of BmBDV-derived transcripts were correlated with the levels of + nsd-2 expression. However, BmBDV-derived transcripts were clearly detected in all parts of the midgut. These results suggest that the infectivity of BmBDV depends mainly on the expression level of + nsd-2 in the midgut and that viral infection is supported even by very faint expression of + nsd-2 . By contrast, the expression levels of + nsd-2 were exceedingly low or undetectable in the middle part of the midgut, indicating that BmBDV infection might occur via another mechanism, independent of + nsd-2 , in the middle part of the midgut. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β superfamily members inhibit Bombyx mori nucleohedrovirus (BmNPV) multiplication in silkworm are not determined. In this study, we first found that BmNPV RNA transcription and protein expression level were regulated by TGF-β members, Decapentaplegic (Bmdpp) and Dawdle (Bmdaw) in the domesticated silkworm, B. mori and silkworm ovary-derived cells. Furthermore, subcellular localization showed that Bmdpp and Bmdaw were mainly presented in cytomembrane of the cultured BmN cells. Tissues expression pattern analysis found that the highest expression levels of Bmdpp and Bmdaw genes were in the hemocyte of fifth instar larvae. During the immune response, the expression level of Bmdpp gene was elevated and Bmdaw gene was declined in BmNPV infected BmN cells and silkworm. The multiplication of BmNPV was inhibited by overexpression of Bmdpp and Bmdaw genes in BmN cells. RNA interference experiments found that the multiplication of BmNPV was raised with specific siRNAs of Bmdpp and Bmdaw genes in BmN cells. The antiviral immune pathways were not significantly regulated by the TGF-β superfamily members. Taken together, these findings provided a clue to understand the function of Bmdpp and Bmdaw gene in response to the BmNPV infection in silkworm. [ABSTRACT FROM AUTHOR]

Viruses and high temperature (HT) are the primary threats to silkworms. Changes in the expression of stress-response genes can be measured using quantitative polymerase chain reaction (qPCR) after exposure to viruses or HT. However, appropriate reference genes (RGs) for qPCR data normalization have not been established in this organism. In this study, we summarized the RGs used in the previous silkworm studies after infection with Bombyx mori nucleopolyhedrovirus (BmNPV), B. mori cytoplasmic polyhedrosis virus (BmCPV), or B. mori densovirus (BmDNV) or after HT treatment. The expression levels of these RGs were extracted from silkworm transcriptome data to screen for candidate RGs that were unaffected by the experimental conditions. Actin-1 ( A1), actin-3 ( A3), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), and translation initiation factor 4a ( TIF- 4A) were selected for further qPCR verification. The results of RNA-seq and qPCR showed that GAPDH and TIF- 4A were suitable RGs after BmNPV challenge or HT stress, whereas TIF- 4A was an appropriate RG for BmCPV or BmDNV-Z challenge in silkworms. These results suggested that TIF-4A may be the most appropriate RG for gene expression analysis after challenge with viruses or HT in silkworms. [ABSTRACT FROM AUTHOR]

Thousands of Hymenopteran endoparasitoids have developed a unique symbiotic relationship with viruses named polydnavirus (PDVs). These viruses immunocompromise the lepidopteran host allowing the survival of the wasp eggs. In a previous work, we have shown the horizontal transfer of some polydnaviral genes into the genome of the Lepidoptera, Spodoptera exigua . One of these genes, BV2-5 (named g asmin ) interferes with actin polymerization, negatively affecting the multiplication of baculovirus in cell culture. In this work, we have focused in the study of the effect of Gasmin expression on different aspects of the baculovirus production. In addition, and since actin polymerization is crucial for phagocytosis, we have studied the effect of Gasmin expression on the larval interaction with bacterial pathogens. Over-expression of Gasmin on hemocytes significantly reduces their capacity to phagocytize the pathogenic bacteria Bacillus thuringiensis . According to these results, gasmin domestication negatively affects baculovirus replication, but increases larvae susceptibility to bacterial infections as pay off. Although the effect of Gasmin on the insect interaction with other pathogens or parasitoids remain unknown, the opposite effects described here could shape the biological history of this species based on the abundance of certain type of pathogens as suggested by the presence of truncated forms of this protein in several regions of the world. [ABSTRACT FROM AUTHOR]

Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2 . IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an “RNAi-of-RNAi” experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17–18) and also not of SBPV (n = 11–12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections. [ABSTRACT FROM AUTHOR]

Genes in the signal transducer and activator of transcription (STAT) family are vital for activities including gene expression and immune response. To investigate the functions of the silkworm Bombyx mori STAT ( Bm-STAT ) gene in antiviral immunity, two Bm-STAT gene isoforms, Bm-STAT -L for long form and Bm-STAT- S for short form, were cloned. Sequencing showed that the open reading frames were 2313 bp encoding 770 amino acid residues for Bm-STAT -L and 2202 bp encoding 734 amino acid residues for Bm-STAT- S. The C-terminal 42 amino acid residues of Bm-STAT-L were different from the last 7 amino acid residues of Bm-STAT-S. Immunofluorescence showed that Bm-STAT was primarily distributed in the nucleus. Transcription levels of Bm-STAT in different tissues were determined by quantitative PCR, and the results revealed Bm-STAT was mainly expressed in testes. Western blots showed two bands with molecular weights of 70 kDa and 130 kDa in testes, but no bands were detected in ovaries by using anti-Bm-STAT antibody as the primary antibody. Expression of Bm-STAT in hemolymph at 48 h post infection with B. mori macula-like virus (BmMLV) was slightly enhanced compared with controls, suggesting a weak response induced by infection with BmMLV. Hemocyte immunofluorescence showed that Bm-STAT expression was elevated in B. mori nucleopolyhedrovirus (BmNPV)-infected cells. Moreover, resistance of BmN cells to BmNPV was reduced by downregulation of Bm-STAT expression and increased by upregulation. Resistance of BmN cells to BmCPV was not significantly improved by upregulating Bm-STAT expression. Therefore, we concluded that Bm-STAT is a newly identified insect gene of the STAT family. The JAK-STAT pathway has a more specialized role in antiviral defense in silkworms, but JAK-STAT pathway is not triggered in response to all viruses. [ABSTRACT FROM AUTHOR]

Spodoptera litura (S. litura) is one of the most destructive agricultural pests worldwide. There is urgent need for a nuclear polyhedrosis virus that is specific to S. litura. To date, there have been no reports regarding the responses of S. litura cells to early Spodoptera litura nucleopolyhedrovirus (SpltNPV) infection due to the lack of a reference genome and transcriptome for S. litura. In this study, a cell transcriptome from the host S. litura was assembled and used for Illumina strand-specific RNA sequencing (RNA-seq) to generate 99180 unigenes, representing the 18 hour infection cycle. More than 2000 S. litura genes were significant differentially regulated throughout the infection. The levels of viral mRNAs began to increase dramatically at 6 hpi, and this increase continued throughout the remainder of the infection. We focused on the expression of genes related to stress responses, apoptosis, metabolic enzymes and host cell innate immune system. A small subset of genes related to host stress response, especially for 62 ones being able to annotated as enzyme, ligand and receptor genes, were observed to be specifically differentially expressed at 6 hpi. At 18 hpi, 104 unigenes were continuously significantly changing from 0 hpi to 18 hpi, considered to be viral multiplication related genes, including 3 annotated SL221 unigenes and 81 viral genes, such as tetraspanin and iap gene. This information and further studies on the regulation of host gene expression by baculovirus infection at early stage will provide the tools needed to enhance the utility of this virus as an effective insecticide. [ABSTRACT FROM AUTHOR]

The prevalence and loads of deformed wing virus (DWV) between honey bee (Apis mellifera L.) colonies from a tropical and a temperate environment were compared. The interaction between these environments and the mite Varroa destructor in relation to DWV prevalence, levels, and overt infections, was also analyzed. V. destructor rates were determined, and samples of mites, adult bees, brood parasitized with varroa mites and brood not infested by mites were analyzed. DWV was detected in 100% of themites and its prevalence and loads in honey bees were significantly higher in colonies from the temperate climate than in colonies from the tropical climate. Significant interactions were found between climate and type of sample, with the highest levels of DWV found in varroa-parasitized brood from temperate climate colonies. Additionally, overt infections were observed only in the temperate climate. Varroa parasitism and DWV loads in bees from colonies with overt infections were significantly higher than in bees from colonies with covert infections. These results suggest that interactions between climate, V. destructor, and possibly other factors, may play a significant role in the prevalence and levels of DWV in honey bee colonies, as well as in the development of overt infections. Several hypotheses are discussed to explain these results. [ABSTRACT FROM AUTHOR]

The sperm of the heteropteran bug Raphigaster nebulosa (Poda) are of two types, differing in length and size of their flagella. The thicker sperm are shorter than the thinner ones and have large mitochondrial derivatives. The presence of virus particles associated with the plasma membrane of thinner sperm is described for the first time; thicker sperm are immune to virus infection. The fact that virus particles are present on thinner sperm only initiates considerations on the transmission of virus. [ABSTRACT FROM AUTHOR]

China has the largest number of managed honey bee colonies globally, but there is currently no data on viral infection in diseased A. mellifera L. colonies in China. In particular, there is a lack of data on chronic bee paralysis virus (CBPV) in Chinese honey bee colonies. Consequently, the present study investigated the occurrence and frequency of several widespread honey bee viruses in diseased Chinese apiaries, and we used the reverse transcription-polymerase chain reaction (RT-PCR) assay. Described was the relationship between the presence of CBPV and diseased colonies (with at least one of the following symptoms: depopulation, paralysis, dark body colorings and hairless, or a mass of dead bees on the ground surrounding the beehives). Phylogenetic analyses of CBPV were employed. The prevalence of multiple infections of honey bee viruses in diseased Chinese apiaries was 100%, and the prevalence of infections with even five and six viruses were higher than expected. The incidence of CBPV in diseased colonies was significantly higher than that in apparently healthy colonies in Chinese A. mellifera aparies, and CBPV isolates from China can be separated into Chinese-Japanese clade 1 and 2. The results indicate that beekeeping in China may be threatened by colony decline due to the high prevalence of multiple viruses with CBPV. [ABSTRACT FROM AUTHOR]

Southern rice black-streaked dwarf virus (SRBSDV) P9-1 is involved in viroplasm formations in the SRBSDV-infected plants and insects. During infection, SRBSDV P9-1 is an important protein. However, the function characterization of P9-1 octamers in vitro and in yeast is still obscure. In this study, the secondary and 3D structure of SRBSDV P9-1 was predicted, then SRBSDV P9-1 was expressed and analyzed in vitro , a size exclusion chromatography assay showed that P9-1 had the ability to form octamers in vitro . Mutational analysis of terminal residues showed that the C-terminal arm (residues 324–347) of P9-1 was importance for the formation of octamers. Furthermore, a yeast two-hybrid assay showed that there were strong interactions between the full-length P9-1s, after deleting the C-terminal arm of P9-1, the interactions between the truncated P9-1s were disappeared in yeast . Collectively, the non-structural protein P9-1 played an essential role in self-interact viroplasm formation in vitro and in yeast , and the C-terminal arm region of P9-1 was important for assembling octamers in vitro . [ABSTRACT FROM AUTHOR]

The potential for infectious pathogens to spillover and emerge from managed populations to wildlife communities is poorly understood, but ecological, evolutionary and anthropogenic factors are all likely to influence the initial exposure and subsequent infection, spread and impact of disease. Fast-evolving RNA viruses, known to cause severe colony losses in managed honeybee populations, deserve particular attention for their propensity to jump between host species and thus threaten ecologically and economically important wild pollinator communities., We review the literature on pollinator viruses to identify biological and anthropogenic drivers of disease emergence, highlight gaps in the literature, and discuss potential management strategies., We provide evidence that many wild pollinator species are exposed to viruses from commercial species, resulting in multiple spillover events. However, it is not clear whether species become infected as a result of spillover or whether transmission is occurring within these wild populations. Ecological traits of pollinating insects, such as overlapping ranges, niches and behaviours, clearly promote cross-species transmission of RNA viruses. Moreover, we conclude that the social behaviour and phylogenetic relatedness of social pollinators further facilitate within- and between-host transmission, leaving these species particularly vulnerable to emerging diseases., We argue that the commercial use of pollinators is a key driver of disease emergence in these beneficial insects and that this must be addressed by management and policy., Synthesis and applications. There are important knowledge gaps, ranging from disease distribution and prevalence, to pathogen life history and virulence, to the impacts of disease emergence, which need to be addressed as research priorities. It is clear that avoiding anthropogenic pathogen spillover is crucial to preventing and managing disease emergence in pollinators, with far-reaching effects on our food security, ecosystem services and biodiversity. We argue that it is crucial to prevent the introduction of diseased pollinators into natural environments, which can be achieved through improved monitoring and management practices. [ABSTRACT FROM AUTHOR]

Populations of honey bees are declining throughout the world, with US beekeepers losing 30% of their colonies each winter. Though multiple factors are driving these colony losses, it is increasingly clear that viruses play a major role. However, information about the molecular mechanisms mediating antiviral immunity in honey bees is surprisingly limited. Here, we examined the transcriptional and epigenetic (DNA methylation) responses to viral infection in honey bee workers. One-day old worker honey bees were fed solutions containing Israeli Acute Paralysis Virus (IAPV), a virus which causes muscle paralysis and death and has previously been associated with colony loss. Uninfected control and infected, symptomatic bees were collected within 20–24 hours after infection. Worker fat bodies, the primary tissue involved in metabolism, detoxification and immune responses, were collected for analysis. We performed transcriptome- and bisulfite-sequencing of the worker fat bodies to identify genome-wide gene expression and DNA methylation patterns associated with viral infection. There were 753 differentially expressed genes (FDR<0.05) in infected versus control bees, including several genes involved in epigenetic and antiviral pathways. DNA methylation status of 156 genes (FDR<0.1) changed significantly as a result of the infection, including those involved in antiviral responses in humans. There was no significant overlap between the significantly differentially expressed and significantly differentially methylated genes, and indeed, the genomic characteristics of these sets of genes were quite distinct. Our results indicate that honey bees have two distinct molecular pathways, mediated by transcription and methylation, that modulate protein levels and/or function in response to viral infections. [ABSTRACT FROM AUTHOR]

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase ( egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (Ld MNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (Ac MNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. Ac MNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant Ac MNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a 'gene for an extended phenotype' is therefore not generally applicable for all baculovirus-host interactions. We hypothesize that in some baculovirus-host systems (including Ld MNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially. [ABSTRACT FROM AUTHOR]

Israeli acute paralysis virus (IAPV) together with Acute bee paralysis virus (ABPV) and Kashmir bee virus (KBV) constitute a complex of closely related dicistroviruses. They are infamous for their high mortality after injection in honeybees. These viruses have also been reported in non- Apis hymenopteran pollinators such as bumblebees, which got infected with IAPV when placed in the same greenhouse with IAPV infected honeybee hives. Here we orally infected Bombus terrestris workers with different doses of either IAPV or KBV viral particles. The success of the infection was established by analysis of the bumblebees after the impact studies: 50 days after infection. Doses of 0.5 × 10 7 and 1 × 10 7 virus particles per bee were infectious over this period, for IAPV and KBV respectively, while a dose of 0.5 × 10 6 IAPV particles per bee was not infectious. The impact of virus infection was studied in micro-colonies consisting of 5 bumblebees, one of which becomes a pseudo-queen which proceeds to lay unfertilized (drone) eggs. The impact parameters studied were: the establishment of a laying pseudo-queen, the timing of egg-laying, the number of drones produced, the weight of these drones and worker mortality. In this setup KBV infection resulted in a significant slower colony startup and offspring production, while only the latter can be reported for IAPV. Neither virus increased worker mortality, at the oral doses used. We recommend further studies on how these viruses transmit between different pollinator species. It is also vital to understand how viral prevalence can affect wild bee populations because disturbance of the natural host-virus association may deteriorate the already critically endangered status of many bumblebee species. [ABSTRACT FROM AUTHOR]

We identified the insect iridovirus IIV-6/CrIV as a pathogen of the cricket Gryllus texensis using electron microscopy (EM) and polymerase chain reaction (PCR) analysis. EM showed that the virus attacks the fat body, an organ important for protein production, immune function and lipid storage. During infection the fat body hypertrophied, but egg production withered, leaving the lateral oviducts empty of eggs; the females were effectively sterile. EM of the testis of infected males suggests that the testis was not invaded by the virus, although sperm taken from the spermatophores of infected males showed little or no motility. Nevertheless, males and females continued to mate when infected. In fact, infected males were quicker to court females than uninfected controls. The virus benefits from the continued sexual behaviour of its host; transmission studies show that the virus can be spread through sexual contact. Sickness behaviour, the adaptive reduction of feeding and sexual behaviour that is induced by an activated immune system, was absent in infected crickets. Total haemolymph protein was reduced, as was phenoloxidase activity, suggesting a reduction in immune protein production by the fat body. The evidence suggests that during IIV-6/CrIV infection, the immune signal(s) that induces sickness behaviour is absent. Curtailment of a host's sickness behaviour may be necessary for any pathogen that is spread by host sexual behaviour [ABSTRACT FROM AUTHOR]

Rice ragged stunt virus (RRSV), an oryzavirus in the family Reoviridae, is transmitted by the brown planthopper, Nilaparvata lugens, in a persistent-propagative manner. Here, we established a continuous cell line of brown planthopper to investigate the mechanism underlying the formation of the viroplasm, the putative site for viral replication and assembly, during infection of RRSV in its insect vector cells. Within 24 h of viral infection of cultured cells, the viroplasm had formed and contained the viral nonstructural proteins Pns6 and Pns10, known to be constituents of viroplasm. Core capsid protein P3, core particles, and newly synthesized viral RNAs were accumulated inside the viroplasm, while outer capsid protein P8 and virions were accumulated at the periphery of the viroplasm, confirming that the viroplasm induced by RRSV infection was the site for viral replication and assembly. Pns10 formed viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix was largely composed of Pns10. Pns6 was recruited in the viroplasm by direct interaction with Pns10. Core capsid protein P3 was recruited to the viroplasm through specific association with Pns6. Knockdown of Pns6 and Pns10 expression using RNA interference inhibited viroplasm formation, virion assembly, viral protein expression, and viral double-stranded RNA synthesis. Thus, the present study shows that both Pns6 and Pns10 of RRSV play important roles in the early stages of viral life cycle in its insect vector cells, by recruiting or retaining components necessary for viral replication and assembly. [ABSTRACT FROM AUTHOR]

Highlights: [•] A complex BTV transmission and maintenance network has emerged in Europe. [•] Wild ruminants are regarded as important actors in this epidemiological network. [•] We suggest that wildlife reservoirs contribute to BTV circulation in the Mediterranean. [•] Research is needed on wildlife–Culicoides and deer–BTV–livestock interactions. [•] Actions in wildlife should focus on monitoring. [ABSTRACT FROM AUTHOR]

Detection of Schmallenberg virus RNA, using real-time RT- PCR, in biting midges ( Culicoides spp.) caught at 48 locations in 2011 and four well-separated farms during 2012 in Denmark, revealed a remarkably rapid spread of virus-infected midges across the country. During 2012, some 213 pools of obsoletus group midges (10 specimens per pool) were examined, and of these, 35 of the 174 parous pools were Schmallenberg virus RNA positive and 11 of them were positive in the heads. Culicoides species-specific PCRs identified both C. obsoletus and C. dewulfi as vectors of Schmallenberg virus. [ABSTRACT FROM AUTHOR]

Some animals change their feeding behaviour when infected with parasites, seeking out substances that enhance their ability to overcome infection. This 'self-medication' is typically considered to involve the consumption of toxins, minerals or secondary compounds. However, recent studies have shown that macronutrients can influence the immune response and that pathogen-challenged individuals can self-medicate by choosing a diet rich in protein and low in carbohydrates. Infected individuals might also reduce food intake when infected (i.e. illness-induced anorexia)., Here, we examine macronutrient self-medication and illness-induced anorexia in caterpillars of the African armyworm ( Spodoptera exempta) by asking how individuals change their feeding decisions over the time course of infection with a baculovirus. We measured self-medication behaviour across several full-sib families to evaluate the plasticity of diet choice and underlying genetic variation., Larvae restricted to diets high in protein ( P) and low in carbohydrate ( C) were more likely to survive a virus challenge than those restricted to diets with a low P : C ratio. When allowed free choice, virus-challenged individuals chose a higher protein diet than controls., Individuals challenged with either a lethal or sublethal dose of virus increased the P : C ratio of their chosen diets. This was mostly due to a sharp decline in carbohydrate intake, rather than an increased intake of protein, reducing overall food intake, consistent with an illness-induced anorexic response. Over time the P : C ratio of the diet decreased until it matched that of controls., Our study provides the clearest evidence yet for dietary self-medication using macronutrients and shows that the temporal dynamics of feeding behaviour depends on the severity and stage of the infection. The strikingly similar behaviour shown by different families suggests that self-medication is phenotypically plastic and not a consequence of genetically based differences in diet choice between families. [ABSTRACT FROM AUTHOR]

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated 'Dak Nong virus (DKNV),' shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae. [ABSTRACT FROM AUTHOR]

Densoviruses are parvoviruses that can be lethal for insects of different orders at larval stages. Although the horizontal transmission mechanisms are poorly known, densoviral pathogenesis usually starts with the ingestion of contaminated food by the host. Depending on the virus, this leads to replication restricted to the midgut or excluding it. In both cases the success of infection depends on the virus capacity to enter the intestinal epithelium. Using the Junonia coenia densovirus (JcDNV) as the prototype virus and the lepidopteran host Spodoptera frugiperda as an interaction model, we focused on the early mechanisms of infection during which JcDNV crosses the intestinal epithelium to reach and replicate in underlying target tissues. We studied the kinetics of interaction of JcDNV with the midgut epithelium and the transport mechanisms involved. Using several approaches, in vivo, ex vivo, and in vitro, at molecular and cellular levels, we show that JcDNV is specifically internalized by endocytosis in absorptive cells and then crosses the epithelium by transcytosis. As a consequence, viral entry disturbs the midgut function. Finally, we showed that four mutations on the capsid of JcDNV affect specific recognition by the epithelial cells but not their binding. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examined the effect of female infection status on male mate choice in Drosophila melanogaster. [•] Males preferentially mated with sham infected females. [•] Sham-infected females had higher reproductive output compared to infected females. [•] We found potential evidence of pre-copulatory male mate choice. [•] We did not find any evidence of post-copulatory male mate choice measured in terms of variation in copulation duration. [ABSTRACT FROM AUTHOR]

Ld652Y cells from the gypsy moth, Lymantria dispar, are highly sensitive to apoptotic stimuli compared to BM-N cells from the silkworm, Bombyx mori, and undergo apoptosis upon infection with various nucleopolyhedroviruses (NPVs). We previously cloned and characterized Dronc homologues from L. dispar Ld652Y and B. mori BM-N cells and demonstrated that L. dispar Dronc (Ld-Dronc) undergoes proteolytic processing more rapidly and extensively than does B. mori Dronc (Bm-Dronc) upon infection with vAc?p35, a p35-defective Autographa californica multiple NPV. Here, we comparatively examined expression and proteolytic processing of the Ld- and Bm-Dronc proteins in the transient expression assays using three different insect cell lines, Ld652Y, BM-N and Spodoptera frugiperda Sf9. We demonstrate that even in the transient expression assays, Ld-Dronc undergoes proteolytic processing and activates caspase-3-like protease more rapidly and extensively than Bm-Dronc in a cell-line independent manner. These results indicate that intrinsic properties of Dronc are involved in the differing capacity of Ld652Y and BM-N cells to induce apoptosis in response to apoptotic stimuli, including NPV infection. [ABSTRACT FROM AUTHOR]

Many species of tsetse flies are infected by a virus that causes salivary gland hypertrophy (SGH) syndrome and the virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies with SGH have a reduced fecundity and fertility. Due to the deleterious impact of SGHV on G. pallidipes colonies, several approaches were investigated to develop a virus management strategy. Horizontal virus transmission is the major cause of the high prevalence of the GpSGHV in tsetse colonies. Implementation of a “clean feeding” regime (fresh blood offered to each set of flies so that there is only one feed per membrane), instead of the regular feeding regime (several successive feeds per membrane), was among the proposed approaches to reduce GpSGHV infections. However, due to the absence of disposable feeding equipment (feeding trays and silicone membranes), the implementation of a clean feeding approach remains economically difficult. We developed a new clean feeding approach applicable to large-scale tsetse production facilities using existing resources. The results indicate that implementing this approach is feasible and leads to a significant reduction in virus load from 109 virus copies in regular colonies to an average of 102.5 and eliminates the SGH syndrome from clean feeding colonies by28 months post implementation of this approach. The clean feeding approach also reduced the virus load from an average of 108 virus copy numbers to an average of 103 virus copies and SGH prevalence of 10% to 4% in flies fed after the clean fed colony. Taken together, these data indicate that the clean feeding approach is applicable in large-scale G. pallidipes production facilities and eliminates the deleterious effects of the virus and the SGH syndrome in these colonies. [ABSTRACT FROM AUTHOR]

The Reverse Transcription-polymerase Chain Reaction (RT-PCR) is an excellent technique for the detection of honeybee viruses. In this study, the presence of Sac Brood Virus (SBV) and Black Queen Cell Virus (BQCV) was demonstrated in 100 samples, collected from Jordanian honeybee colonies by employing RT-PCR. The collected samples represented infected (depopulation, paralysis, or dark coloring), dead, and apparently healthy honeybees of different developmental stages (adult and larvae). SBV was detected in 37% of the samples, whereas BQCV was detected in 5%. Nucleotide sequences of the PCR products from each virus was determined and found to be 433 and 309 nucleotides in SBV and BQCV, respectively. The identities to the Gene Bank were 95% for SBV and 91% for BQCV. This is the first record of BQCV in Jordan. [ABSTRACT FROM AUTHOR]

Abstract: Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24–48hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins. [Copyright &y& Elsevier]

Abstract: Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 103 copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees. [Copyright &y& Elsevier]

Abstract: Cucumber mosaic virus, Tomato spotted wilt virus, Tomato mosaic virus, Tomato chlorosis virus, Pepino mosaic virus, Torrado tomato virus and Tomato infectious chlorosis virus cause serious damage and significant economic losses in tomato crops worldwide. The early detection of these pathogens is essential for preventing the viruses from spreading and improving their control. In this study, a procedure based on two multiplex RT-PCRs was developed for the sensitive and reliable detection of these seven viruses. Serial dilutions of positive controls were analysed by this methodology, and the results were compared with those obtained by ELISA and singleplex versions of RT-PCR. The multiplex and singleplex RT-PCR assays were able to detect specific targets at the same dilution and were 100 times more sensitive than ELISA. The multiplex versions were able to detect composite samples containing different concentrations of specific targets at ratios from 1:1 to 1:1000. In addition, 45 symptomatic tomato samples collected in different tomato-growing areas of Sicily (Italy) were analysed by multiplex RT-PCR, singleplex RT-PCR and commercially available ELISA tests. Similar results were obtained using the RT-PCR techniques, with a higher sensitivity than ELISA, revealing a common occurrence of mixed infections and confirming the presence of these seven virus species in Italy. [Copyright &y& Elsevier]

Abstract: Insects are infected by a variety of pathogens, including bacteria, fungi and viruses, which have been studied largely for their potential as biocontrol agents, but are also important in insect conservation (biodiversity) and as model systems for other diseases. Whilst the dynamics of host–pathogen interactions are well-studied at the population level, less attention has been paid to the critical within-host infection stage. Here, the reproductive rate of the pathogen is largely determined by how it exploits the host; the resources supplied by the host in terms of size and condition; competition with other pathogens; and the speed with which it kills the host (death being an inevitable outcome for obligate-killing pathogens). In this paper we aim to build upon recent developments in the literature by conducting single infection bioassays to obtain data on growth and fitness parameters for phenotypically different and similar strains of nucleopolyhedroviruses in the Lepdipoteran host Spodoptera exigua. Using these data, a simple mechanistic mathematical model (a coupled system of differential equations) is derived, fitted and parameter sensitivity predictions are made which support empirical findings. We unexpectedly found that initial growth of virus within the host occurs at a double-exponential rate, which contrasts with empirical findings for vertebrate host–pathogen systems. Moreover, these infection rates differ between strains, which has significant implications for the evolution of virulence and strain coexistence in the field, which are still relative unknowns. Furthermore, our model predicts that, counter to intuition, increased viral doses may lead to a decrease in viral yield, which is supported by other studies. We explain the mechanism for this phenomenon and discuss its implications for insect host–pathogen ecology. [Copyright &y& Elsevier]

Abstract: Here we report the characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate, named SfMNPV-6nd, that does not cause the liquefaction of the host integument. The sequencing of the chitinase A (v-chiA) gene from SfMNPV-6nd revealed that it had a frameshift mutation that greatly reduced size of the putative enzyme. In order to evaluate the suitability of SfMPNV-6nd as a biopesticide, this isolate was compared with the highly virulent SfMNPV-19. Our results showed that the LC50 of the two isolates were not significantly different, but that SfMNPV-6nd took a longer period of time to kill second instar S. frugiperda than SfMNPV-19. [Copyright &y& Elsevier]