Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii.

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

Transcriptome Analysis Reveals a Diverse Range of Novel Viruses in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, Soldier flies (Inopus spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

Orthoflavivirus Lammi in Russia: Possible Transovarial Transmission and Trans-Stadial Survival in Aedes cinereus (Diptera, Culicidae).

In the last few years, there has been a dramatic increase in the number of discovered viruses that are transmitted by arthropods. Some of them are pathogenic for humans and mammals, and the pathogenic potential of others is unknown. The genus Orthoflavivirus belongs to the family Flaviviridae and includes arboviruses that cause severe human diseases with damage to the central nervous system and hemorrhagic fevers, as well as viruses with unknown vectors and viruses specific only to insects. The latter group includes Lammi virus, first isolated from a mosquito pool in Finland. It is known that Lammi virus successfully replicates in mosquito cell lines but not in mammalian cell cultures or mice. Lammi virus reduces the reproduction of West Nile virus during superinfection and thus has the potential to reduce the spread of West Nile virus in areas where Lammi virus is already circulating. In this work, we isolated Lammi virus from a pool of adult Aedes cinereus mosquitoes that hatched from larvae/pupae collected in Saint Petersburg, Russia. This fact may indicate transovarial transmission and trans-stadial survival of the virus.

Assessment of larval and pupal indices of dengue mosquito vectors in a North-Eastern state of Tripura, India.

BACKGROUND OBJECTIVES: Dengue is a major vector-borne disease having public health importance. It is caused by Dengue Virus (DENV) and is transmitted by mosquitoes of Aedes species. With the unavailability of a vaccine, vector control remains the only preventive measure for dengue. Studies have already been conducted to establish the presence of dengue vectors in the north-eastern states of India. However, limited studies have been conducted in Tripura state. In the present study we aimed to identify the preferred breeding habitats of dengue vectors in the state. METHODS: Clinical case data of dengue since the last five years was studied and the areas with the highest case numbers were identified. Entomological investigation was carried out in areas reporting the highest number of cases. Larvae were collected from the breeding habitats using standard protocol followed by morphological and molecular identification. Further, House index (HI), Container index (CI) and Pupal index (PI) were determined. The positive pools were then processed for incrimination for the presence of dengue virus. Calculation of entomological indices was done. RESULTS: Of the total 815 containers searched, 36.80% containers were positive for mosquito larvae. Among the immature mosquito collection, 836 adults emerged and were identified as Aedes albopictus using standard taxonomic keys followed by molecular methods. HI, CI and PI, varied from 15.38% to 100%, 21% to 31.04 %, and 2.93% to 110.53% respectively. However, none of the pools was positive for dengue virus. INTERPRETATION CONCLUSION: The present study identified Ae. albopictus as a potential vector of dengue in Tripura. The study gave important insights on the preferred larval habitats and provides information on the indication of displacement of Ae. albopictus from rural to urban and semi-urban areas. However, longitudinal studies for longer time frame are necessary for any conclusive remarks.

Assessment of Mayaro virus vector competence of the mosquito Aedes aegypti (Linnaeus, 1762) populations in Argentine using dose-response assays.

Mayaro virus (MAYV; Alphavirus: Togaviridae) is an emerging pathogen in Latin America, causing fever and polyarthritis. Sporadic outbreaks of MAYV have occurred in the region, with reported human cases being imported to Europe and North America. Although primarily a risk for those residing in the Amazon basin's tropical forests, recent reports highlight that urbanization would increase the risk of MAYV transmission in Latin America. Urban emergence depends on human susceptibility and the ability of mosquitos like Aedes aegypti  (Linnaeus, 1762) (Diptera: Culicidae) to transmit MAYV. Despite the absence of active MAYV transmission in Argentine, the risk of introduction is substantial due to human movement and the presence of Ae. aegypti in the region. This study aimed to evaluate the susceptibility of different Argentine Ae. aegypti populations to MAYV genotype L (MAYV-L) using dose-response assays and determine barriers to virus infection, dissemination and transmission. Immature mosquito stages were collected in Buenos Aires, Córdoba and Rosario cities. Female Ae. aegypti (F2) were orally infected by feeding on five concentrations of MAYV-L, ranging from 1.0 to 6.0 log10 PFU/mL. Abdomens, legs and saliva were analysed using viral plaque assays. Results revealed that MAYV-L between infection and dissemination were associated with viral doses rather than the population origin. Infection rates varied between 3% and 65%, with a 50% infectious dose >5.5 log10 PFU/mL. Dissemination occurred at 39%, with a 50% dissemination dose of ~6.0 log10 PFU/mL. Dissemination among infected mosquitoes ranged from 60% to 86%, and transmission from disseminated mosquitoes ranged from 11% to 20%. Argentine Ae. aegypti populations exhibited a need for higher viral doses of MAYV-L than those typically found in humans to become infected. In addition, only a small proportion of infected mosquitoes were capable of transmitting the virus. Understanding MAYV transmission in urban areas is crucial for public health interventions.

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems.

Clostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%-92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry. IMPORTANCE: Clostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

Compatibility of the fungus Beauveria bassiana and Trichoplusia ni SNPV against the cabbage looper Trichoplusia ni: crop plant matters.

BACKGROUND: Microbial insecticides are an important weapon in insect pest management, but their use is still relatively limited. One approach for increasing their efficacy and use could be to combine different pathogens to increase pest mortality. However, little is known about whether increasing pathogen diversity will improve pest management. Here, we investigated the compatibility of two pathogens for the management of the cabbage looper, Trichoplusia ni, T. ni nucleopolyhedrovirus (TniSNPV) and the entomopathogenic fungus Beauveria bassiana, on two crops, tomato and broccoli. The pathogens were applied to individual plants using ultra low volume sprays, alone or in combination, either synchronously or asynchronously. Healthy third-instar T. ni larvae were introduced to the plants before application and collected by destructive sampling 24 h after the last pathogen application. RESULTS: Combined applications did not result in an increase in larval mortality compared to TniSNPV alone, although mortality was generally high. B. bassiana was considerably less effective on broccoli compared to tomato. In both the combined treatments, virus-induced mortality was approximately 50% lower when applied together with the fungus, while fungus-induced mortality was not affected by the virus, even when the virus was introduced 24 h before the fungus. CONCLUSION: While our results suggest that applying this combination of entomopathogens would not be beneficial for pest management, this study illustrates the need to consider the target crop as an important driver of the efficacy of both single and mixed pathogen applications in the field. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Bmo-miR-6498-5p suppresses Bombyx mori nucleopolyhedrovirus infection by down-regulating BmPLPP2 to modulate pyridoxal phosphate content in B. mori.

The RNA interference pathway mediated by microRNAs (miRNAs) is one of the methods to defend against viruses in insects. Recent studies showed that miRNAs participate in viral infection by binding to target genes to regulate their expression. Here, we found that the Bombyx mori miRNA, miR-6498-5p was down-regulated, whereas its predicted target gene pyridoxal phosphate phosphatase PHOSPHO2 (BmPLPP2) was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Both in vivo and in vitro experiments showed that miR-6498-5p targets BmPLPP2 and suppresses its expression. Furthermore, we found miR-6498-5p inhibits BmNPV genomic DNA (gDNA) replication, whereas BmPLPP2 promotes BmNPV gDNA replication. As a pyridoxal phosphate (PLP) phosphatase (PLPP), the overexpression of BmPLPP2 results in a reduction of PLP content, whereas the knockdown of BmPLPP2 leads to an increase in PLP content. In addition, exogenous PLP suppresses the replication of BmNPV gDNA; in contrast, the PLP inhibitor 4-deoxypyridoxine facilitates BmNPV gDNA replication. Taken together, we concluded that miR-6498-5p has a potential anti-BmNPV role by down-regulating BmPLPP2 to modulate PLP content, but BmNPV induces miR-6498-5p down-regulation to promote its proliferation. Our findings provide valuable insights into the role of host miRNA in B. mori-BmNPV interaction. Furthermore, the identification of the antiviral molecule PLP offers a novel perspective on strategies for preventing and managing viral infection in sericulture.

Single-Nucleus Sequencing of Silkworm Larval Brain Reveals the Key Role of Lysozyme in the Antiviral Immune Response in Brain Hemocytes.

INTRODUCTION: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown. METHODS: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain. RESULTS: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects. CONCLUSION: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

LIMITED ACCUMULATION AND PERSISTENCE OF AN INFLUENZA A VIRUS IN TADPOLE SNAILS (PHYSA SPP.).

Waterfowl infected with avian influenza A viruses (IAVs) shed infectious virus into aquatic environments, providing a mechanism for transmission among waterfowl, while also exposing the entire aquatic ecosystem to the virus. Aquatic invertebrates such as freshwater snails are likely exposed to IAVs in the water column and sediment. Freshwater snails comprise a significant portion of some waterfowl species' diets, so this trophic interaction may serve as a novel route of IAV transmission. In these experiments, tadpole snails (Physa spp.) were exposed to a low-pathogenicity IAV (H3N8) to determine whether snails can accumulate the virus and, if so, how long virus persists in snail tissues. Snail tissues were destructively sampled and tested by reverse-transcription quantitative real-time PCR. Our experiments demonstrated that tadpole snails do accumulate IAV RNA in their tissues, although at low titers, for at least 96 h. These results indicate that it may be possible for IAV transmission to occur between waterfowl via ingestion of a natural invertebrate prey item; however, the time frame for transmission may be limited.

Host ranges of Sf-rhabdoviruses harbored by lepidopteran insects and insect cell lines.

Cell lines derived from Spodoptera frugiperda (Sf), which are the most widely used hosts in the baculovirus-insect cell system, are contaminated with Sf-rhabdoviruses (Sf-RVs). In this study, we identified a closely related virus (Sf-CAT-RV) in the caterpillar species used to isolate the original Sf cell line. We then evaluated the Sf-RV and Sf-CAT-RV host ranges, found Sf-CAT-RV could infect Vero cells, and obtained results suggesting both variants can infect mouse ear fibroblasts. In addition, we found both variants could establish pantropic infections in severely immunocompromised (RAG2/IL2RG-/-) mice. However, both variants were cleared by two weeks post-inoculation and neither produced any symptoms or obvious adverse outcomes in these hosts. We conclude the caterpillars used to isolate Sf21 cells were the most likely source of the Sf-RV contaminant, Sf-RVs and their Sf-CAT-RV progenitor have broader host ranges than expected from previous work, but neither variant poses a serious threat to human health.

An Ascovirus Utilizes Different Types of Host Larval Regulated Cell Death Mechanisms To Produce and Release Vesicles.

Ascoviruses are insect-specific viruses that are thought to utilize the cellular apoptotic processes of host larvae to produce numerous virion-containing vesicles. In this study, we monitored the in vivo infection processes of Heliothis virescens ascovirus 3h (HvAV-3h) to illustrate the regulated cell death (RCD) of host cells. Transmission electron microscopic observations did not reveal any morphological markers of apoptosis in the fat bodies or hemocytes of HvAV-3h-infected Helicoverpa armigera or Spodoptera exigua larvae. However, several hemocytes showed the morphological criteria for necrosis and/or pyroptosis. Further in vitro biochemical tests were performed to confirm the RCD type of host cells after infection with HvAV-3h. Different morphological characteristics were found between the early (prior to 24 hours post-infection, [hpi]) and later (48 to 120 hpi) stages in both HvAV-3h infected larval fat bodies and hemocytes. In the early stages, the virions could only be found in several adipohemocytes, and the fat bodies were cleaving their contained lipid inclusions into small lipid dots. In the later stage, both fat bodies and hemocytes were filled with numerous virions. According to the morphological characteristics of HvAV-3h infected larval fat bodies or hemocytes, the pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, and the systematic pathogenic mode of ascovirus infection was refined in this study. This study details the complete infection process of ascoviruses, which provides insights into the relationship between a pathogenesis of an insect virus and the RCD of different host tissues at different stages of infection. IMPORTANCE Viruses and other pathogens can interrupt host cellular apoptosis to gain benefits, such as sufficient resources and a stable environment that enables them to complete their replication and assembly. It is unusual for viruses to code proteins with homology to caspases, which are commonly recognized as apoptosis regulators. Ascoviruses are insect viruses with special cytopathology, and they have been hypothesized to induce apoptosis in their host larvae via coding a caspase-like protein. This enables them to utilize the process of cellular apoptosis to facilitate vesicle formation and replication. However, our previous studies revealed different trends. The fat bodies and hemocytes of Heliothis virescens ascovirus 3h (HvAV-3h)-infected larvae did not show any morphological markers of apoptosis but did display necrosis and/or pyroptosis morphological characteristics. The pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, which can help us understand the relationship between the pathogenesis of an insect virus and host RCD.

Endogenous Retroviruses Augment Amphibian (Xenopus laevis) Tadpole Antiviral Protection.

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

Sustained Ranavirus Outbreak Causes Mass Mortality and Morbidity of Imperiled Amphibians in Florida.

A persistent 2-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and disease signs in N. perstriatus continued for 5 weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a frog virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

Microbial Composition in Larval Water Enhances Aedes aegypti Development but Reduces Transmissibility of Zika Virus.

Arthropod-borne viruses comprise a significant global disease burden. Surveillance and mitigation of arboviruses like Zika virus (ZIKV) require accurate estimates of transmissibility by vector mosquitoes. Although Aedes species mosquitoes are established as competent ZIKV vectors, differences in experimental protocols across studies prevent direct comparisons of relative transmissibility. An understudied factor complicating these comparisons is differential environmental microbiota exposures, where most vector competence studies use mosquitoes reared in laboratory tap water, which does not represent the microbial complexity of environmental water where wild larvae develop. We simulated natural larval development by rearing Californian Aedes aegypti larvae with microbes obtained from cemetery headstone water compared to conventional tap water. A. aegypti larvae reared in environmental cemetery water pupated 3 days faster and at higher rates. Mosquitoes reared in environmental water were less competent vectors of ZIKV than laboratory water-reared A. aegypti, as evidenced by significantly reduced infection and transmission rates. Microbiome comparisons of laboratory water- and environment water-reared mosquitoes and their rearing water showed significantly higher bacterial diversity in environment water. Despite this pattern, corresponding differences in bacterial diversity were not consistently observed between the respective adult mosquitoes. We also observed that the microbial compositions of adult mosquitoes differed more by whether they ingested a bloodmeal than by larval water type. Together, these results highlight the role of transient microbes in the larval environment in modulating A. aegypti vector competence for ZIKV. Laboratory vector competence likely overestimates the true transmissibility of arboviruses like ZIKV when conventional laboratory water is used for rearing. IMPORTANCE We observed that A. aegypti mosquitoes reared in water from cemetery headstones instead of the laboratory tap exhibited a reduced capacity to become infected with and transmit Zika virus. Water from the environment contained more bacterial species than tap water, but these bacteria were not consistently detected in adult mosquitoes. Our results suggest that rearing mosquito larvae in water collected from local environments as opposed to laboratory tap water, as is conventional, could provide a more realistic assessment of ZIKV vector competence since it better recapitulates the natural environment in which larvae develop. Given that laboratory vector competence is used to define the species to target for control, the use of environmental water to rear larvae could better approximate the microbial exposures of wild mosquitoes, lessening the potential for overestimating ZIKV transmission risk. These studies raise the question of whether rearing larvae in natural water sources also reduces vector competence for other mosquito-borne viruses.

Circulating dengue virus serotypes and vertical transmission in Aedes larvae during outbreak and inter-outbreak seasons in a high dengue risk area of Sri Lanka.

BACKGROUND: Spatial and temporal changes in the dengue incidence are associated with multiple factors, such as climate, immunity among a population against dengue viruses (DENV), circulating DENV serotypes and vertical transmission (VT) of DENV in an area at a given time. The level of VT in a specific location has epidemiological implications in terms of viral maintenance in vectors. Identification of the circulating DENV serotypes in both patients and Aedes mosquito larvae in an area may be useful for the early detection of outbreaks. We report here the results of a prospective descriptive study that was conducted to detect the levels of VT in Aedes mosquito larvae and circulating DENV serotypes in patients and Aedes mosquito larvae from December 2015 to March 2017 in an area of Sri Lanka at high risk for dengue. METHODS: A total of 200 patients with clinically suspected dengue who had been admitted to a tertiary care hospital during a dengue outbreak (3 study periods: December 2015-January 2016, June-August 2016, December 2016-January 2017) and in the inter-outbreak periods (February-May 2016 and September-November 2016) were investigated. Blood samples were drawn from the study participants to test for DENV. The houses of the study participants were visited within 7 days of admission to the hospital, and Aedes larvae were also collected within a radius of 400 m from the houses. The larvae were separately identified to species and then pooled according to each patient's identification number. Patients' sera and the Aedes larvae were tested to identify the infecting DENV serotypes using a reverse transcription PCR (RT-PCR) method. Levels of VT in Aedes mosquito larvae were also identified. RESULTS: All four DENV serotypes (DENV-1 to -4) were identified in the study area. In the early part of the study (December 2015-February 2016), DENV-3 was predominant and from April 2016 to March 2017, DENV-2 became the most predominant type. Four cases of DENV co-infections were noted during the study period in patients. Interestingly, all four DENV serotypes were detected in Aedes albopictus larvae, which was the prominent immature vectorial form identified throughout the study period in the area, showing 9.8% VT of DENV. With the exception of DENV-4, the other three DENV serotypes were identified in Aedes aegypti larvae with a VT of 8.1%. CONCLUSION: Comparatively high rates of VT of DENV was detected in Ae. albopictus and Ae. aegypti larvae. A shift in the predominant DENV serotype with simultaneous circulation of all four DENV serotypes was identified in the study area from December 2015 to March 2017.

The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.