Ecological features and insecticide resistance of Aedes albopictus in Xi'an, a high-risk dengue transmission area in China.

Background: Dengue, a mosquito-borne viral disease, has occurred in many cities in China, and it tends to spread to higher latitudes (Huang et al., 2023). Xi'an, situated in central-west China, has witnessed an increase imported cases in the past few years, raising concerns of local dengue transmission. It is crucial to investigate the population density of Aedes albopictus and its insecticides resistance to enhance early warning of dengue fever. Methods: Eight sampling sites in eight counties (YT, BL, WY, CH, YL, LN, LT, ZZ) of Xi'an city were surveyed by larval dipping and human-baited double net trap biweekly from June 2021 to September 2022. The Breteau Index (BI, number of positive containers per 100 houses) and Container Index (CI, the percentage of containers containing larvae or pupae) were used to assess larval density, and the human-baited double net trap (HDN, the number of Ae. albopictus females collected per person per hour) to indicate human bating rate (HBR). Meanwhile, the association between the meteorological factors and mosquito density was analyzed. The Ae. albopictus adult insecticides resistance was evaluated by the World Health Organization (WHO) standard resistance bioassay. Adult females were exposed to insecticide-impregnated paper for 1 h, then transferred to the recovery tube, and mortality rate was calculated after 24 h. According to the Implementation Plan for National Vector Surveillance (2016), resistance status was classified into three levels based on mortality: <80% mortality as resistant, between 80% and 98% mortality as possibly resistant, and >98% mortality as sensitive. Results: From June 2021 to September 2022, a total of 1,065 houses were surveyed for water holding containers, and 99 of 430 water holding containers were checked to be positive for Ae. albopictus larvae and pupae. A total of 1,048 Ae. albopictus females were collected. The average BI, CI and HBR were 10.39, 21.41, and 11.20 female/man/hour in 2021 and 8.86, 20.86, and 11.63 f/m/h in 2022, respectively. The findings showed that the BI exceeded 5 in most months and reached above 20 in specific months. The CI varied in different months and monitoring sites, with the highest CI in August 2021 and July 2022. The discarded tires had the highest positivity rate, with up to 40.32% testing positive for Ae. albopictus larvae. The monthly average temperature showed a positive correlation with CI (r = 0.77), and the monthly BI was positively associated with CI (r = 0.93). The BI, CI, and HBR were significantly higher in the rainy season than other seasons. The bioassay results showed that the mortality rate of Ae. albopictus at the YT monitoring site was 76.92%, indicating resistance to deltamethrin. The mortality rate of Ae. Albopictus at BL, WY, CH, YL, LN, LT, and ZZ sampling sites were varying from 81.25%∼100%, suggesting possibly resistant or still sensitive to beta-cypermethrin, alpha-cypermethrin, malathion, chlorpyrifos, and propoxur.

Israeli Acute Paralysis Virus Is an Emerging Pathogen Contributing to Brood Disease of Apis cerana.

Larval mortality is the primary symptom of diseased Apis cerana colonies, often attributed to sacbrood virus (SBV) and Melissococcus plutonius. However, the impact of other common honeybee viruses is frequently overlooked, and their pathogenicity to A. cerana remains poorly understood. To investigate the causes of the increasing disease incidence in A. cerana brood, we conducted an epidemiological survey, collecting 70 samples from 19 sites across nine provinces in China. Furthermore, we examined the pathogenicity of Israeli acute paralysis virus (IAPV) in A. cerana brood through artificial inoculation experiments. Our results demonstrate that, besides SBV and M. plutonius, the infection rate and viral load of IAPV in diseased brood are significantly high. Brood artificially inoculated with high concentrations of IAPV exhibited a significant increase in mortality and displayed clinical symptoms similar to those observed in naturally infected colonies. Moreover, a limited resistance to IAPV was observed in A. cerana brood, with some individuals able to restrict viral proliferation. Our study highlights the previously unrecognized pathogenicity of IAPV to A. cerana brood, demonstrating that IAPV poses a significant threat similar to SBV and M. plutonius. We emphasize that IAPV should be recognized as an emerging pathogen causing brood disease in A. cerana and managed accordingly in beekeeping practices.

Larval crowding enhances dengue virus loads in Aedes aegypti, a relationship that might increase transmission in urban environments.

BACKGROUND: Climate change and urbanization will alter the global distribution of disease vectors, changing the disease burden in yet unpredictable ways. Aedes aegypti is a mosquito responsible for transmitting dengue, Zika, chikungunya, and yellow fever viruses that breeds in containers associated with urban environments. We sought to understand how ambient temperature and larval densities in the immature aquatic phases determine adult life history traits and dengue virus loads post-infection. We predicted that larval crowding and high temperatures would both lead to smaller mosquitoes that might struggle to invest in an immune response and, hence, would exhibit high viral loads. METHODS: We first examined larval densities from urban and rural areas via a meta-analysis. We then used these data to inform a laboratory-based 2x2 design examining the interacting effects of temperature (21 vs. 26°C) and density (0.2 vs. 0.4 larvae/mL) on adult life history and dengue virus loads. RESULTS: We found that urban areas had an ~8-fold increase in larval densities compared to more rural sites. In the lab, we found that crowding had more impact on mosquito traits than temperature. Crowding led to slower development, smaller mosquitoes, less survival, lower fecundity, and higher viral loads, as predicted. The higher temperature led to faster development, reduced fecundity, and lower viral loads. The virus-reducing effect of higher temperature rearing was, however, overwhelmed by the impact of larval crowding when both factors were present. CONCLUSIONS: These data reveal complex interactions between the environmental effects experienced by immature mosquitoes and adult traits. They especially highlight the importance of crowding with respect to adult viral loads. Together, these data suggest that urban environments might enhance dengue virus loads and, therefore, possibly transmission, a concerning result given the increasing rates of urbanization globally.

Functional regulation of microRNA-184 in the replication and infection of Autographa californica multiple nucleopolyhedrovirus.

MicroRNAs (miRNAs) represent a class of short, non-coding RNAs that are widely acknowledged as crucial participants in virus-host interactions. MiR-184, a highly conserved and abundant miRNA in insects, has yet to be extensively studied for its involvement in baculovirus infection. In this study, we investigated how miR-184 affects the infection and replication of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The results indicated that after AcMNPV infection, there was an initial increase in the expression of miR-184 within 24 h, followed by a subsequent decrease. MiR-184 can inhibit AcMNPV's DNA replication and budded virus production by directly targeting four viral genes, namely ie1, ac66, p49, and lef9. Moreover, suppressing miR-184 expression enhanced the insecticidal efficacy of AcMNPV against Spodoptera exigua larvae and markedly elevated the host ATPase gene expressions. These findings showed that miR-184 had a substantial impact on the interactions between baculoviruses and insects, presenting a prospective candidate for developing highly effective miRNA-based biopesticides.

Assessment of housekeeping genes stability for gene transcription regulation analysis of Spodoptera littoralis (Lepidoptera: Noctuidae) under Spodoptera littoralis nucleopolyhedrovirus viral infection.

BACKGROUND: Normalization with respect to stable housekeeping genes is important to facilitate gene transcription regulation research and acquire more accurate quantitative polymerase chain reaction (qPCR) data. In the current study, five candidates housekeeping genes of the cotton leafworm, Spodoptera littoralis encoding for Actin (Actin), elongation factor 1-alpha (EF1α), ribosomal protein S3 (RPS3), ribosomal protein 49 (RP49), and Ubiquitin (Ubi), were evaluated as normalization housekeeping genes under Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) viral infection. METHODS AND RESULTS: The qPCR results confirmed the expression of all five housekeeping genes in S. littoralis viral infected larvae. The expression profiles of the housekeeping genes showed that the EF1α, Actin, and RP49 had the minimum average Ct values of 18.41 ± 0.66, 18.84 ± 0.90 and 19.01 ± 0.87 in all infected samples, respectively. While RPS3 and Ubi showed the maximum average Ct of 21.61 ± 0.51 and 21.11 ± 0.82, respectively. According to the results of ΔCt and geNorm analysis, EF1α was ranked as the most stable housekeeping gene during infection time-course. While by using BestKeeper, geNorm and NormFinder, the Ubi, RP49, and RPS3 showed the most genes transcription stability. The obtained results were also validated using the Cytochrome c oxidase (COX) gene transcripts in response to SpliNPV infection. CONCLUSIONS: The results revealed that EF1α and Ubi were the most stable housekeeping genes to be used for normalizing S. littoralis gene transcription regulation under SpliNPV infection. These findings, provide a significant addition for gene transcription regulation studies of S. littoralis upon infection using SpliNPV as a bio-agent.

Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly.

The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

Host Jump of an Exotic Fish Rhabdovirus into a New Class of Animals Poses a Disease Threat to Amphibians.

Spring viremia of carp virus (SVCV) is a rhabdovirus that primarily infects cyprinid finfishes and causes a disease notifiable to the World Organization for Animal Health. Amphibians, which are sympatric with cyprinids in freshwater ecosystems, are considered non-permissive hosts of rhabdoviruses. The potential host range expansion of SVCV in an atypical host species was evaluated by testing the susceptibility of amphibians native to the Pacific Northwest. Larval long-toed salamanders Ambystoma macrodactylum and Pacific tree frog Pseudacris regilla tadpoles were exposed to SVCV strains from genotypes Ia, Ib, Ic, or Id by either intraperitoneal injection, immersion, or cohabitation with virus-infected koi Cyprinus rubrofuscus. Cumulative mortality was 100% for salamanders injected with SVCV, 98-100% for tadpoles exposed to virus via immersion, and 0-100% for tadpoles cohabited with SVCV-infected koi. Many of the animals that died exhibited clinical signs of disease and SVCV RNA was found by in situ hybridization in tissue sections of immersion-exposed tadpoles, particularly in the cells of the gastrointestinal tract and liver. SVCV was also detected by plaque assay and RT-qPCR testing in both amphibian species regardless of the virus exposure method, and viable virus was detected up to 28 days after initial exposure. Recovery of infectious virus from naïve tadpoles cohabited with SVCV-infected koi further demonstrated that SVCV transmission can occur between classes of ectothermic vertebrates. Collectively, these results indicated that SVCV, a fish rhabdovirus, can be transmitted to and cause lethal disease in two amphibian species. Therefore, members of all five of the major vertebrate groups (mammals, birds, reptiles, fish, and amphibians) appear to be vulnerable to rhabdovirus infections. Future research studying potential spillover and spillback infections of aquatic rhabdoviruses between foreign and domestic amphibian and fish species will provide insights into the stressors driving novel interclass virus transmission events.

Larval Competition between Aedes and Culex Mosquitoes Carries over to Higher Arboviral Infection during Their Adult Stage.

The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major threat as vectors for dengue, chikungunya (CHIKV), and Japanese encephalitis virus (JEV). These mosquitoes share a similar ecological niche as larvae, but the carry-over effects of aquatic larval interactions to the terrestrial adult stage remain largely unknown and their medical relevance requires further investigation. This study examines the context dependency of larval interactions among Aedes albopictus, Aedes japonicus, and Culex pipiens. The survival, development time, growth, and energetic storage were measured in different European populations within density-response (intraspecific) experiments and replacement (interspecific) experiments at 20 °C and 26 °C. Overall, Ae. japonicus was the weakest competitor, while competition between Ae. albopictus and Cx. pipiens varied with temperature. Adults emerging from this larval competition were infected as follows: Culex pipiens with WNV, Ae. albopictus with CHIKV, and Ae. japonicus with JEV. While no JEV infection was observed, mosquitoes experiencing interspecific interactions during their larval stages exhibited higher infection rates and viral RNA titers for CHIKV and WNV. This increased susceptibility to viral infection after larval competition suggests a higher risk of arbovirus transmission in co-occurring populations.

Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles.

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For the past 4 years, American insect-rearing facilities have experienced a distinctive larval pathology and colony collapse of farmed Zophobas morio (superworm). By means of cryo-EM, we discovered the causative agent: a densovirus that we named Zophobas morio black wasting virus (ZmBWV). We confirmed the etiology of disease by fulfilling Koch's postulates and characterizing strains from across the United States. ZmBWV is a member of the family Parvoviridae with a 5,542 nt genome, and we describe intersubunit interactions explaining its expanded internal volume relative to human parvoviruses. Cryo-EM structures at resolutions up to 2.1 Å revealed single-strand DNA (ssDNA) ordering at the capsid inner surface pinned by base-binding pockets in the capsid inner surface. Also, we demonstrated the prophylactic potential of non-pathogenic strains to provide cross-protection in vivo.

Dose-dependent dynamics of densovirus infection in two nymphalid butterfly species utilizing native or exotic host plants.

Insects are attacked by a diverse range of microbial pathogens in the wild. In herbivorous species, larval host plants frequently play a critical role in mediating susceptibility to infection. Characterizing such plant-mediated effects on herbivore-pathogen interactions can provide insight into patterns of infection across wild populations. In this study, we investigated the effects of host plant use by two North American butterflies, Euphydryas phaeton (Nymphalidae) and Anartia jatrophae (Nymphalidae), on entomopathogen infection across a range of three doses. Both of these herbivores recently incorporated the same exotic plant, Plantago lanceolata (Plantaginaceae), into their host range and are naturally infected by the same entomopathogen, Junonia coenia densovirus (Parvoviridae), in wild populations. We performed two factorial experiments in which E. phaeton and A. jatrophae were reared on either P. lanceolata or a native host plant [Chelone glabra (Plantaginaceae) for E. phaeton; Bacopa monnieri (Plantaginaceae) for A. jatrophae] and inoculated with either a low, medium, or high dose of the virus. In E. phaeton, the outcomes of infection were highly dose-dependent, with inoculation with higher viral doses resulting in faster time to death and greater mortality. However, neither survival nor postmortem viral burdens varied depending upon the host plant that was consumed. In contrast, host plant use had a strong effect on viral burdens in A. jatrophae, with consumption of the exotic plant appearing to enhance host resistance to infection. Together, these results illustrate the variable influences of host plant use on herbivore resistance to infection, highlighting the importance of investigating plant-herbivore relationships within a tritrophic framework.

Utilizing Zebrafish Embryos for Replication of Tulane Virus: A Human Norovirus Surrogate.

The zebrafish larvae/embryo model has been shown to support the replication of seven strains (G1.7[P7], GII.2[P16], GII.3[P16], GII.4[P4], GII.4[P16], GII.6[P7], and GII.17[P13]) of human norovirus (HuNoV). However, due to challenges in consistently obtaining HuNoV-positive stool samples from clinical sources, evaluating HuNoV surrogates in this model is highly valuable. This study assesses the potential of zebrafish embryos and larvae as a model for Tulane virus (TuV) replication. Three infection methods were examined: microinjection, immersion, and feeding. Droplet digital PCR was used to quantify viral RNA across all three infection methods. Microinjection of 3 nL of TuV into zebrafish embryos (< 6-h post-fertilization) resulted in significant replication, with viral RNA levels reaching 6.22 logs at 4-day post-infection. In contrast, the immersion method showed no replication after immersing 4-day post-fertilization (dpf) larvae in TuV suspension for 6 h. Similarly, no replication was observed with the feeding method, where Paramecium caudatum loaded with TuV were fed to 4 dpf larvae. The findings indicate that the zebrafish embryo model supports TuV replication through the microinjection method, suggesting that TuV may serve as a useful surrogate for studying HuNoV pathogenesis. Additionally, TuV can be utilized in place of HuNoV in method optimization studies using the zebrafish embryo model, circumventing the limited availability of HuNoV.

Prevalence and molecular analysis of some important viruses in honey bee colonies in Türkiye: the status of multiple infections.

In this study, seven bee viruses of significant importance for bee health in Türkiye were investigated using one-step RT-PCR. For this purpose, larvae from 1183 hives and adult bees from 1196 hives were sampled from 400 apiaries in 40 provinces. The prevalence of viral infections in hives was as follows: acute bee paralysis virus (ABPV), 6.4%; black queen cell virus (BQCV), 77%; chronic bee paralysis virus (CBPV), 3.2%; deformed wing virus (DWV), 63.8%; Israel acute bee paralysis virus (IAPV), 7%; Kashmir bee virus (KBV), 2.7%; sacbrood virus (SBV), 49.7%. Moreover, 50 different combinations of viral infections were identified in the hives. While dual infections (36.1%) were the most common in hives, triple infections with BQCV, DWV, and SBV were found to have the highest prevalence (22.1%). At least one viral infection was detected in all of the apiaries tested. Phylogenetic analysis showed that the isolates from this study generally exhibited the highest similarity to previously reported Turkish isolates. When similarity ratios and the locations and types of amino acid mutations were analyzed, it was observed that the isolates from our study exhibited high similarity to isolates from various countries, including China, the United Kingdom, Syria, and Germany.

Tomato Spotted Wilt Virus Suppresses the Antiviral Response of the Insect Vector, Frankliniella occidentalis, by Elevating an Immunosuppressive C18 Oxylipin Level Using Its Virulent Factor, NSs.

Orthotospovirus tomatomaculae (tomato spotted wilt virus, TSWV) is transmitted by the western flower thrips, Frankliniella occidentalis. Epoxyoctadecamonoenoic acids (EpOMEs) function as immune-suppressive factors, particularly in insects infected by viral pathogens. These oxylipins are produced by cytochrome P450 monooxygenases (CYPs) and are degraded by soluble epoxide hydrolase (sEH). In this study, we tested the hypothesis that TSWV modulates the EpOME level in the thrips to suppress antiviral responses and enhance its replication. TSWV infection significantly elevated both 9,10-EpOME and 12,13-EpOME levels. Following TSWV infection, the larvae displayed apoptosis in the midgut along with the upregulated expression of four caspase genes. However, the addition of EpOME to the viral treatment notably reduced apoptosis and downregulated caspase gene expressions, which led to a marked increase in TSWV titers. The CYP and sEH genes of F. occidentalis were identified, and their expression manipulation using RNA interference (RNAi) treatments led to significant alternations in the insect's immune responses and TSWV viral titers. To ascertain which viral factor influences the host EpOME levels, specialized RNAi treatments targeting genes encoded by TSWV were administered to larvae infected with TSWV. These treatments demonstrated that NSS expression is pivotal in manipulating the genes involved in EpOME metabolism. These results indicate that NSs of TSWV are crucially linked with the elevation of host insect EpOME levels and play a key role in suppressing the antiviral responses of F. occidentalis.

Pathogen occurrence in different developmental stages of the invasive Vespa velutina nigrithorax (Buysson, 1905).

BACKGROUND: The yellow-legged hornet (Vespa velutina nigrithorax) is a predatory species native to South-East Asia. The hornet is invasive in Europe, spreading to several countries and becoming a pest for Apis mellifera due to its behaviour of preying in front of apiaries. The aim of this study was (i) to investigate the presence of honey bee pathogens within the developmental stages of V. velutina after neutralizing a nest in Bologna province (Emilia-Romagna, Italy) and (ii) to analyze the mitochondrial DNA to determine if the population derived from the population initially introduced in Europe. RESULTS: The results indicated that deformed wing virus (82.76%) and Nosema ceranae (67.28%) were the most prevalent pathogens. Deformed wing virus, N. ceranae and sacbrood virus were found in all investigated stages, while chronic bee paralysis virus and Kashmir bee virus were exclusively found in foraging adults. All detected viruses were found to be replicative, highlighting active infection in the hosts. The mtDNA analysis demonstrated that the origin derived from the invasive population arrived in France. CONCLUSION: This study underscores the importance of further research to understand the effect of interspecific transmission, especially concerning the potential role of these pathogens as a biocontrol for the invasive V. velutina nigrithorax. © 2024 Society of Chemical Industry.

AcMNPV-miR-2 affects Autographa californica nucleopolyhedrovirus infection by regulating the expression of ac28 and several other viral early genes.

Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes ac28 (lef-6), ac37 (lef-11), ac49, and ac63. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration. IMPORTANCE: Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.

Environmental DNA concentrations vary greatly across productive and degradative conditions, with implications for the precision of population estimates.

Population size is an important metric to inform the conservation and management of species. For aquatic species, environmental DNA (eDNA) concentration has been suggested for non-invasively estimating population size. However, many biotic and abiotic factors simultaneously influence the production and degradation of eDNA which can alter the relationship between population size and eDNA concentration. We investigated the influence of temperature, salinity, and ranavirus infection on eDNA concentrations using tadpole mesocosms. Using linear regression models, we tested the influence of each experimental treatment on eDNA concentrations at three time points before and during epidemics. Prior to infection, elevated temperatures lowered eDNA concentrations, indicating that degradation was the driving force influencing eDNA concentrations. During early epidemics, no treatments strongly influenced eDNA concentrations and in late epidemics, productive forces dominated as ranavirus intensity and dead organisms increased eDNA concentrations. Finally, population size was only an important predictor of eDNA concentration in late epidemics and we observed high levels of variation between samples of replicate mesocosms. We demonstrate the complexities of several interacting factors influencing productive and degradative forces, variation in influences on eDNA concentration over short time spans, and examine the limitations of estimating population sizes from eDNA with precision in semi-natural conditions.

Bombyx mori UFL1 facilitates BmNPV proliferation by regulating host cell apoptosis through PERK.

Ubiquitin-fold modifier 1 (UFM1) is attached to protein substrates through the sequential activity of an E1 (UBA5)-E2 (UFC1)-E3 (UFL1) cascade. UFL1 is the E3 ligase for UFMylation in vertebrates. However, there have been no studies on UFL1 in silkworm to date. In this study, we identified a UFL1 ortholog in Bombyx mori genome. Spatio-temporal expression profiles showed that BmUFL1 expression was high in the midgut, epidermis, and testis and in the pupa-adult stage. BmUFL1 knockdown inhibited B. mori nucleopolyhedrovirus (BmNPV) proliferation, while BmUFL1 overexpression promoted BmNPV proliferation. Mechanically, protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling and cell apoptosis are involved in BmUFL1-regulated BmNPV proliferation. Overall, these results suggest that BmUFL1 facilitates BmNPV proliferation in silkworm.

Protocol for linking enhanced interferon immunity to virus resistance in gene-knockout zebrafish.

A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for determining the change in interferon response by microinjecting plasmids into one-cell-stage zebrafish embryos. We describe steps for comparing the resistance potential to virus infection in wild-type and knockout zebrafish larvae following plasmid microinjection. We then detail how to link the enhanced interferon immunity to the improved resistance in knockout zebrafish larvae by gene-rescue experiments. For complete details on the use and execution of this protocol, please refer to Qu et al.1.

Morphological, Biological, and Molecular Characterization of Type I Granuloviruses of Spodoptera frugiperda.

Granuloviruses (GVs) Betabaculovirus associated with the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), especially those of the type I, have scarcely been studied. These GVs might be an effective alternative for the biocontrol of this insect. In this study, the native GVs SfGV-CH13 and SfGV-CH28 were isolated from FAW larvae and characterized for morphology, molecular traits, and insecticidal activity. The elapsed time between symptomatic infection of larvae and stop feeding as well as the weight of larvae before death or prior to pupation were also evaluated. Both GVs had ovoid shape and a length of 0.4 µm. They had the same DNA restriction profiles and their genome sizes were about 126 kb. The symptomatic infection with the tested GVs mainly caused flaccidity of larva body and discoloration of integument. The integument lysis was only observed in 8% of infected larvae. Infected larvae gradually stopped feeding. Overall, these symptoms are characteristic of infections caused by type I GVs, which are known as monoorganotropic or slow-killing GVs. The median lethal dose (LD50) values for SfGV-CH13 and SfGV-CH28 isolates were 5.4 × 102 and 1.1 × 103 OBs/larva, respectively. The median lethal time (LT50) ranged from 17 to 24 days. LT50 values decreased as the viral dose was increased. The elapsed time from symptomatic infection until pupation and body weight of larvae (third instar) were higher with SfGV-CH28 than SfGV-CH13. Both granulovirus isolates were able to kill the FAW larvae from the 12th day.

Transcriptome analysis of CpGV in midguts of type II resistant codling moth larvae and identification of contaminant infections by SNP mapping of RNA-Seq data.

Various isolates of the Cydia pomonella granulovirus (CpGV) are used as insect pest control agents against codling moth (CM, Cydia pomonella L.), a predominant pest in apple orchards. Three different types (I-III) of dominantly inherited field resistance of CM larvae to CpGV have been recently identified. In this study, transcription of virus genes in midgut cells of type II-resistant CM larvae infected with different CpGV isolates, i.e., CpGV-M and CpGV-S (both prone to type II resistance) as well as CpGV-E2 (breaking type II resistance) was determined by strand-specific RNA sequencing (RNA-Seq) at an early infection stage (72 h post infection). Based on principal component analysis of read counts and the quantitative distribution of single nucleotide polymorphisms (SNPs) in the RNA-Seq data, a bioinformatics analysis pipeline was developed for an a posteriori identification of the infective agents. We report that (i) identification of infective agent is crucial, especially in in vivo infection experiments, when activation of covert virus infections is a possibility, (ii) no substantial difference between CpGV-M and CpGV-S transcription was found in type II-resistant CM larvae despite a different resistance mechanism, (iii) the transcription level of CpGV-M and CpGV-S was much lower than that of CpGV-E2, and (iv) orf59 (sod), orf89 (pif-6), orf92 (p18), and orf137 (lef-10) were identified as significantly downregulated genes in resistance-prone isolates CpGV-M and CpGV-S. For type II resistance of CM larvae, we conclude that CpGV-M and CpGV-S are both able to enter midgut cells, but viral transcription is significantly impaired at an early stage of infection compared to the resistance-breaking isolate CpGV-E2. IMPORTANCE: CpGV is a highly virulent pathogen of codling moth, and it has been developed into one of the most successful commercial baculovirus biocontrol agents for pome fruit production worldwide. The emergence of field resistance in codling moth to commercial CpGV products is a threat toward the sustainable use of CpGV. In recent years, different types of resistance (type I-III) were identified. For type II resistance, very little is known regarding the infection process. By studying the virus gene expression patterns of different CpGV isolates in midguts of type II-resistant codling moth larvae, we found that the type II resistance mechanism is most likely based on intracellular factors rather than a receptor component. By applying SNP mapping of the RNA-Seq data, we further emphasize the importance of identifying the infective agents in in vivo experiments when activation of a covert infection cannot be excluded.