Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Genetic determinants of antiviral immunity in dipteran insects - Compiling the experimental evidence.

The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission.

Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction.

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.

Sensing Viral Infections in Insects: A Dearth of Pathway Receptors.

Insects, the most diverse group of animals, can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses can be transmitted to humans, while bee and silkworm viruses cause important economic losses. Like all invertebrates, insects rely solely on innate immunity to counter viral infections. Protein-based mechanisms, involving restriction factors and evolutionarily conserved signaling pathways regulating transcription factors of the NF-kB and STAT families, participate in the control of viral infections in insects. In addition, RNA-based responses play a major role in the silencing of viral RNAs. We review here our current state of knowledge on insect antiviral defense mechanisms, which include conserved as well as adaptive, insect-specific strategies. Identification of the innate immunity receptors that sense viral infection in insects remains a major challenge for the field.

Mosquito-Specific Viruses-Transmission and Interaction.

Mosquito-specific viruses (MSVs) are a subset of insect-specific viruses that are found to infect mosquitoes or mosquito derived cells. There has been an increase in discoveries of novel MSVs in recent years. This has expanded our understanding of viral diversity and evolution but has also sparked questions concerning the transmission of these viruses and interactions with their hosts and its microbiome. In fact, there is already evidence that MSVs interact with the immune system of their host. This is especially interesting, since mosquitoes can be infected with both MSVs and arthropod-borne (arbo) viruses of public health concern. In this review, we give an update on the different MSVs discovered so far and describe current data on their transmission and interaction with the mosquito immune system as well as the effect MSVs could have on an arboviruses-co-infection. Lastly, we discuss potential uses of these viruses, including vector and transmission control.

Bombyx mori bidensovirus infection alters the intestinal microflora of fifth instar silkworm (Bombyx mori) larvae.

Bacterial diseases can occur as a result of disruption of the intestinal microbial population in the silkworm, Bombyx mori, and are often induced by bidensovirus (BmBDV) infection. We investigated the effects of BmBDV infection on intestinal microbes and immune gene responses in fifth instar silkworm larvae. Midgut contents were collected from BmBDV-infected and uninfected silkworms at 48, 96, and 144 h post-infection (hpi) and the intestinal flora were analyzed using 16S rRNA gene Illumina MiSeq sequencing technology. The abundance of intestinal bacteria differed between BmBDV-infected and uninfected silkworms. There were no significant differences in bacterial diversity at 48 and 96 hpi, but bacterial diversity in infected larvae was lower at 144 hpi compared with that of uninfected larvae. At the phylum level, the ratio of Proteobacteria was higher in infected larvae than in uninfected larvae at 48 and 96 hpi, but was lower after 144 hpi, while the ratio of Firmicutes had increased relative to uninfected silkworms. At the genus level, the ratio of Enterococcus increased gradually in infected silkworms, however, proportion of bacteria genera Incertae sedis were increased at 96 hpi, and the proportion of Lactococcus had decreased at 96 hpi. Principal component analysis showed that the proportion of Enterococcus species present was negatively correlated with most dominant genera. Increases in the abundances of the genera Anderseniella, Simplicispira, Enterococcus and, genera Incertae sedis, were associated with BmBDV infection. Quantitative reverse transcription-polymerase chain reaction indicated that expression levels of genes associated with immune deficiency (IMD), Toll, and JAK/STAT pathways were higher at 144 hpi with BmBDV infection. Enterococcus abundance was higher and was positively correlated with the expression level of spatzle-1, peptidoglycan recognition protein LE, and peptidoglycan recognition protein LB genes, suggesting that an increase in the abundance of Enterococcus leads to activation of the Toll and IMD immune pathways.

Inhibitory effects of viral infection on cancer development.

Immune responses evoked on viral infections prevent the dissemination of infection that otherwise leads to the development of diseases in host organisms. In the present study, we investigated whether viral infection influences tumorigenesis in cancer-bearing animals using a Drosophila model of cancer. Cancer was induced in the posterior part of wing imaginal discs through the simultaneous inhibition of apoptosis and cell-cycle checkpoints. The larvae and embryos of cancer-induced flies were infected with Drosophila C virus, a natural pathogen to Drosophila, and larval wing discs and adult wings were morphologically examined for cancer characteristics relative to uninfected controls. We found that viral infections brought about an approximately 30% reduction in the rate of cancer development in both wing discs and wings. These inhibitory effects were not observed when growth-defective virus was used to infect animals. These results indicate that productive viral infections repress tumorigenesis in Drosophila.

Coevolution of hytrosaviruses and host immune responses.

BACKGROUND: Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160-174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV's specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV's asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. RESULTS: Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host's immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly's RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. CONCLUSIONS: The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive 'explorers' to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

Increased antiviral capacity of transgenic silkworm via knockdown of multiple genes on Bombyx mori bidensovirus.

Bombyx mori bidensovirus (BmBDV) causes fatal flacherie disease leading to severe economic losses in sericultures. The BmDNV-Z genome contains two single-stranded DNA molecules, VD1 and VD2. For generating silkworm lines with antiviral properties, two transgenic RNA interference (RNAi) vectors were constructed. Open reading frames (ORFs) 1-4 of VD1 were knockdown by vector pb-BDV1 while ORF1a, ORF1b, and ORF3 of VD2 were knockdown by vector pb-BDV2. Transgenic silkworm lines BDV1-I and BDV2-I were generated via RNAi microinjection. Mortality rates of BDV1-I and BDV2-I were reduced by 45% and 39%, respectively, and quantitative PCR showed that VD1 and VD2 contents in BDV1-I and BDV2-I were significantly lower than in the non-transgenic line. However, economic traits showed no obvious differences. Thus, knockdown of multiple BmDNV-Z genes provides strong resistance to BDV1-I and BDV2-I lines, and these can be used in sericulture without hampering silk production.

Biotechnological Applications of an Insect-Specific Alphavirus.

The coupling of viral and arthropod host diversity, with evolving methods of virus discovery, has resulted in the identification and classification of a growing number of novel insect-specific viruses (ISVs) that appear to be evolutionarily related to many human pathogens but have either lost or have yet to gain the ability to replicate in vertebrates. The discovery of ISVs has raised many questions as to the origin and evolution of many human pathogenic viruses and points to the role that arthropods may play in this evolutionary process. Furthermore, the use of ISVs to control the transmission of arthropod-borne viruses has been proposed and demonstrated experimentally. Previously, our laboratory reported on the discovery and characterization of Eilat virus (EILV), an insect-specific alphavirus that phylogenetically groups within the mosquito-borne clade of medically relevant alphaviruses, including eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV), as well as chikungunya virus (CHIKV). Despite its evolutionary relationship to these human pathogens, EILV is unable to replicate in vertebrate cells due to blocks at attachment/entry and RNA replication. We recently demonstrated that, using a chimeric virus approach, EILV could be utilized as a platform for vaccine and diagnostic development, serving as a proof-of-concept for other ISVs. Due to the vast abundance of ISVs, there is an untapped resource for the development of vaccines and diagnostics for a variety of human pathogens and further work in this area is warranted.

iTRAQ-based quantitative proteomics analysis of molecular mechanisms associated with Bombyx mori (Lepidoptera) larval midgut response to BmNPV in susceptible and near-isogenic strains.

Bombyx mori nucleopolyhedrovirus (BmNPV) has been identified as a major pathogen responsible for severe economic loss. Most silkworm strains are susceptible to BmNPV, with only a few highly resistant strains thus far identified. Here we investigated the molecular basis of silkworm resistance to BmNPV using susceptible (the recurrent parent P50) and resistant (near-isogenic line BC9) strains and a combination of iTRAQ-based quantitative proteomics, reverse-transcription quantitative PCR and Western blotting. By comparing the proteomes of infected and non-infected P50 and BC9 silkworms, we identified 793 differentially expressed proteins (DEPs). By gene ontology and KEGG enrichment analyses, we found that these DEPs are preferentially involved in metabolism, catalytic activity, amino sugar and nucleotide sugar metabolism and carbon metabolism. 114 (14.38%) DEPs were associated with the cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. After removing the genetic background and individual immune stress response proteins, we identified 84 DEPs were found that are potentially involved in resistance to BmNPV. Further studies showed that a serine protease was down-regulated in P50 and up-regulated in BC9 after BmNPV infection. Taken together, these results provide insights into the molecular mechanism of silkworm response to BmNPV. BIOLOGICAL SIGNIFICANCE: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, causing serious losses in sericulture every year. However, the molecular mechanisms of BmNPV infection and host defence remain unclear. Here we combined quantitative proteomic, bioinformatics, RT-qPCR and Western blotting analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut, and that these changes are related to cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. Five important differentially expression proteins were validation by independent approaches. These finding will help address the molecular mechanisms of silkworm resistance to BmNPV and provide a molecular target for resisting BmNPV.

A chikungunya fever vaccine utilizing an insect-specific virus platform.

Traditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. To overcome these tradeoffs, we developed the insect-specific alphavirus, Eilat virus (EILV), as a vaccine platform. To address the chikungunya fever (CHIKF) pandemic, we used an EILV cDNA clone to design a chimeric virus containing the chikungunya virus (CHIKV) structural proteins. The recombinant EILV/CHIKV was structurally identical at 10 Å to wild-type CHIKV, as determined by single-particle cryo-electron microscopy, and it mimicked the early stages of CHIKV replication in vertebrate cells from attachment and entry to viral RNA delivery. Yet the recombinant virus remained completely defective for productive replication, providing a high degree of safety. A single dose of EILV/CHIKV produced in mosquito cells elicited rapid (within 4 d) and long-lasting (>290 d) neutralizing antibodies that provided complete protection in two different mouse models. In nonhuman primates, EILV/CHIKV elicited rapid and robust immunity that protected against viremia and telemetrically monitored fever. Our EILV platform represents the first structurally native application of an insect-specific virus in preclinical vaccine development and highlights the potential application of such viruses in vaccinology.

Immune defence mechanisms of triatomines against bacteria, viruses, fungi and parasites.

Triatomines are vectors that transmit the protozoan haemoflagellate Trypanosoma cruzi, the causative agent of Chagas disease. The aim of the current review is to provide a synthesis of the immune mechanisms of triatomines against bacteria, viruses, fungi and parasites to provide clues for areas of further research including biological control. Regarding bacteria, the triatomine immune response includes antimicrobial peptides (AMPs) such as defensins, lysozymes, attacins and cecropins, whose sites of synthesis are principally the fat body and haemocytes. These peptides are used against pathogenic bacteria (especially during ecdysis and feeding), and also attack symbiotic bacteria. In relation to viruses, Triatoma virus is the only one known to attack and kill triatomines. Although the immune response to this virus is unknown, we hypothesize that haemocytes, phenoloxidase (PO) and nitric oxide (NO) could be activated. Different fungal species have been described in a few triatomines and some immune components against these pathogens are PO and proPO. In relation to parasites, triatomines respond with AMPs, including PO, NO and lectin. In the case of T. cruzi this may be effective, but Trypanosoma rangeli seems to evade and suppress PO response. Although it is clear that three parasite-killing processes are used by triatomines - phagocytosis, nodule formation and encapsulation - the precise immune mechanisms of triatomines against invading agents, including trypanosomes, are as yet unknown. The signalling processes used in triatomine immune response are IMD, Toll and Jak-STAT. Based on the information compiled, we propose some lines of research that include strategic approaches of biological control.

Immune signaling pathways activated in response to different pathogenic micro-organisms in Bombyx mori.

The JAK/STAT, Toll, Imd, and RNAi pathways are the major signaling pathways associated with insect innate immunity. To explore the different immune signaling pathways triggered in response to pathogenic micro-organism infections in the silkworm, Bombyx mori, the expression levels of the signal transducer and activator of transcription (BmSTAT), spatzle-1 (Bmspz-1), peptidoglycan-recognition protein LB (BmPGRP-LB), peptidoglycan-recognition protein LE (BmPGRP-LE), argonaute 2 (Bmago2), and dicer-2 (Bmdcr2) genes after challenge with Escherichia coli (E. coli), Serratiamarcescens (Sm), Bacillus bombyseptieus (Bab), Beauveriabassiana (Beb), nucleopolyhedrovirus (BmNPV), cypovirus (BmCPV), bidensovirus (BmBDV), or Nosemabombycis (Nb) were determined using real-time PCR. We found that the JAK/STAT pathway could be activated by challenge with BmNPV and BmBDV, the Toll pathway could be most robustly induced by challenge with Beb, the Imd pathway was mainly activated in response to infection by E. coli and Sm, and the RNAi pathway was not activated by viral infection, but could be triggered by some bacterial infections. These findings yield insights into the immune signaling pathways activated in response to different pathogenic micro-organisms in the silkworm.

Host adaptation to viruses relies on few genes with different cross-resistance properties.

Host adaptation to one parasite may affect its response to others. However, the genetics of these direct and correlated responses remains poorly studied. The overlap between these responses is instrumental for the understanding of host evolution in multiparasite environments. We determined the genetic and phenotypic changes underlying adaptation of Drosophila melanogaster to Drosophila C virus (DCV). Within 20 generations, flies selected with DCV showed increased survival after DCV infection, but also after cricket paralysis virus (CrPV) and flock house virus (FHV) infection. Whole-genome sequencing identified two regions of significant differentiation among treatments, from which candidate genes were functionally tested with RNAi. Three genes were validated--pastrel, a known DCV-response gene, and two other loci, Ubc-E2H and CG8492. Knockdown of Ubc-E2H and pastrel also led to increased sensitivity to CrPV, whereas knockdown of CG8492 increased susceptibility to FHV infection. Therefore, Drosophila adaptation to DCV relies on few major genes, each with different cross-resistance properties, conferring host resistance to several parasites.

Previous exposure to an RNA virus does not protect against subsequent infection in Drosophila melanogaster.

BACKGROUND: Immune priming has been shown to occur in a wide array of invertebrate taxa, with individuals exposed to a pathogen showing increased protection upon subsequent exposure. However, the mechanisms underlying immune priming are poorly understood. The antiviral RNAi response in Drosophila melanogaster is an ideal candidate for providing a specific and acquired response to subsequent infection. We exposed D. melanogaster to two challenges of a virus known to produce an antiviral RNAi response, to examine whether any protective effects of prior exposure on survival were observed. RESULTS: In this experiment we found no evidence that prior exposure to Drosophila C Virus (DCV) protects flies from a subsequent lethal challenge, with almost identical levels of mortality in flies previously exposed to DCV or a control. CONCLUSIONS: Our results confirm the finding that 'acquired' immune responses are not ubiquitous across all invertebrate-pathogen interactions. We discuss why we may have observed no effect in this study, with focus on the mechanistic basis of the RNAi pathway.

Analysis of the structural proteins from the Musca domestica hytrosavirus with an emphasis on the major envelope protein.

The Musca domestica hytrosavirus (MdHV), a member of the family Hyrosaviridae, is a large, dsDNA, enveloped virus that infects adult house flies and causes a diagnostic hypertrophy of the salivary gland. Herein, studies were directed at identifying key structural components of the viral envelope and nucleocapsid. SDS-PAGE of detergent-treated virus fractions identified protein bands unique to the envelope and nucleocapsid components. Using prior LC-MSMS data we identified the viral ORF associated with the major envelope band, cloned and expressed recombinant viral antigens, and prepared a series of polyclonal sera. Western blots confirmed that antibodies recognized the target viral antigen and provided evidence that the viral protein MdHV96 underwent post-translational processing; antibodies bound to the target high molecular weight parent molecule as well as distinct sets of smaller bands. Immuno gold electron microscopy demonstrated that the anti-MdHV96 sera recognized target antigens associated with the envelope. The nucleocapsids migrated from the virogenic stroma in the nucleus through the nuclear membrane into the cytoplasm, where they acquired an initial envelope that contained MdHV96. This major envelope protein, appeared to incorporate into intracellular membranes of both the caniculi and rough endoplasmic reticulum membranes and mediate binding to the nucleocapsids. Oral infection bioassays demonstrated that the anti-HV96 polyclonal sera acted as neutralizing agents in suppressing the levels of orally acquired infections.

[New genotype of the sacbrood virus of the honeybee Apis mellifera].

The sacbrood virus (SBV) leads to death of honeybee larvae. Until now, there were known three SBV genotypes: European, Asian, and South African (E. Grabensteiner et al., 2001; S. E. Choe et al., 2012). Serologic assay, sequencing and phylogenetic analysis of the SBV RNA-polymerase gene fragments resulted in discovery of two genotypes of SBV circulating among the honeybee Apis mellifera in the European region of Russian Federation (RF). One of them forms a new distinct genetic lineage of SBV (genotype 4), which has not been described before and has an intermediate position between the Asian and the South African genotypes on a phylogenetic tree. The viruses belonging to this group were isolated from honeybee in Kaluga region in 1986, and in 2006 from honeybee introduced earlier to Moscow from Uzbekistan. The sequence homology inside this group is 94.2%, whereas this one between different groups is not higher then 80.6-83.5%. Another group represents the European genotype of SBV circulating in Krasnodar Territory, Adyghe Republic, and Moscow region. Two SBV genotypes from Russian Federation can be differentiated using PCR and radial immunodiffusion test (RID). As for RID, both genotypes react with antiserum #6(1), but only the fourth genotype reacts with antiserum #58.

Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies.

The health of the honeybee and, indirectly, global crop production are threatened by several biotic and abiotic factors, which play a poorly defined role in the induction of widespread colony losses. Recent descriptive studies suggest that colony losses are often related to the interaction between pathogens and other stress factors, including parasites. Through an integrated analysis of the population and molecular changes associated with the collapse of honeybee colonies infested by the parasitic mite Varroa destructor, we show that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season. The de-stabilisation of DWV infection is associated with an immunosuppression syndrome, characterized by a strong down-regulation of the transcription factor NF-κB. The centrality of NF-κB in host responses to a range of environmental challenges suggests that this transcription factor can act as a common currency underlying colony collapse that may be triggered by different causes. Our results offer an integrated account for the multifactorial origin of honeybee losses and a new framework for assessing, and possibly mitigating, the impact of environmental challenges on honeybee health.

Regulation of Drosophila-virus interaction.

Drosophila melanogaster is a useful model system for deciphering mammalian biological processes including development, innate immunity and cancer. Most genetic studies conducted in Drosophila have focused on the immune response against microbial infection and the results obtained have been extrapolated to other organisms. During the last decade the issue of the antiviral response attracted a great deal of interest. In this review we highlight recent discoveries in the role of RNA interference pathway in antiviral response in Drosophila with a focus on the role of miRNAs as both host defense elements and helpers of viral replication.