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.

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.

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.

Histone H2A Nuclear/Cytoplasmic Trafficking Is Essential for Negative Regulation of Antiviral Immune Response and Lysosomal Degradation of TBK1 and IRF3.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

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.

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.

Natural Coinfection between Novel Species of Baculoviruses in Spodoptera ornithogalli Larvae.

Spodoptera ornithogalli (Guenée) (Lepidoptera: Noctuidae) is an important pest in different crops of economic relevance in America. For its control, strategies that include chemicals are usually used; so, the description of entomopathogens would be very useful for the formulation of biopesticides. In this regard, two different baculoviruses affecting S. ornithogalli were isolated in Colombia, with one of them being an NPV and the other a GV. Ultrastructural, molecular, and biological characterization showed that both isolates possess the 38 core genes and are novel species in Baculoviridae, named as Spodoptera ornithogalli nucleopolyhedrovirus (SporNPV) and Spodoptera ornithogalli granulovirus (SporGV). The bioassays carried out in larvae of S. ornithogalli and S. frugiperda showed infectivity in both hosts but being higher in the first. In addition, it was observed that SporGV potentiates the insecticidal action of SporNPV (maximum value in ratio 2.5:97.5). Both viruses are individually infective but coexist in nature, producing mixed infections with a synergistic effect that improves the performance of the NPV and enables the transmission of the GV, which presents a slowly killing phenotype.

Detection of Jingmenviruses in Japan with Evidence of Vertical Transmission in Ticks.

Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.

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.

First Record of Natural Transovarial Transmission of Dengue Virus in Aedes albopictus from Cuba.

Transovarial transmission (TOT) of dengue virus (DENV) in Aedes spp. is an important mechanism for DENV maintenance in nature and may be important in initiating outbreaks. The objective of this study was to explore the occurrence of TOT in wild Aedes albopictus populations in Cuba. Mosquito larvae were collected in Cotorro municipality, Havana, Cuba, and identified to species. Fifteen pools of Ae. albopictus each containing 30 larvae were processed for DENV detection by using conventional reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. Four out of 15 pools processed were positive for DENV-3, but no other DENV serotype was detected. This is the first time TOT of DENV detected in Cuban field populations of Ae. albopictus, and this suggests that this species may be an important vector of DENV in Cuba.

High Temperature Cycles Result in Maternal Transmission and Dengue Infection Differences Between Wolbachia Strains in Aedes aegypti.

Environmental factors play a crucial role in the population dynamics of arthropod endosymbionts, and therefore in the deployment of Wolbachia symbionts for the control of dengue arboviruses. The potential of Wolbachia to invade, persist, and block virus transmission depends in part on its intracellular density. Several recent studies have highlighted the importance of larval rearing temperature in modulating Wolbachia densities in adults, suggesting that elevated temperatures can severely impact some strains, while having little effect on others. The effect of a replicated tropical heat cycle on Wolbachia density and levels of virus blocking was assessed using Aedes aegypti lines carrying strains wMel and wAlbB, two Wolbachia strains currently used for dengue control. Impacts on intracellular density, maternal transmission fidelity, and dengue inhibition capacity were observed for wMel. In contrast, wAlbB-carrying Ae. aegypti maintained a relatively constant intracellular density at high temperatures and conserved its capacity to inhibit dengue. Following larval heat treatment, wMel showed a degree of density recovery in aging adults, although this was compromised by elevated air temperatures. IMPORTANCE In the past decades, dengue incidence has dramatically increased all over the world. An emerging dengue control strategy utilizes Aedes aegypti mosquitoes artificially transinfected with the bacterial symbiont Wolbachia, with the ultimate aim of replacing wild mosquito populations. However, the rearing temperature of mosquito larvae is known to impact on some Wolbachia strains. In this study, we compared the effects of a temperature cycle mimicking natural breeding sites in tropical climates on two Wolbachia strains, currently used for open field trials. When choosing the Wolbachia strain to be used in a dengue control program it is important to consider the effects of environmental temperatures on invasiveness and virus inhibition. These results underline the significance of understanding the impact of environmental factors on released mosquitoes, in order to ensure the most efficient strategy for dengue control.

Starvation at the larval stage increases the vector competence of Aedes aegypti females for Zika virus.

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small 'Starve' and large 'Control' Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus.

Ovitrap surveillance of dengue vector mosquitoes in Bandung City, West Java Province, Indonesia.

Larval surveillance is the central approach for monitoring dengue vector populations in Indonesia. However, traditional larval indices are ineffective for measuring mosquito population dynamics and predicting the dengue transmission risk. We conducted a 14-month ovitrap surveillance. Eggs and immature mosquitoes were collected on a weekly basis from an urban village of Bandung, namely Sekejati. Ovitrap-related indices, namely positive house index (PHI), ovitrap index (OI), and ovitrap density index (ODI), were generated and correlated with environmental variables, housing type (terraced or high-density housing), ovitrap placement location (indoor or outdoor; household or public place), and local dengue cases. Our results demonstrated that Aedes aegypti was significantly predominant compared with Aedes albopictus at each housing type and ovitrap placement location. Ovitrap placement locations and rainfall were the major factors contributing to variations in PHI, OI, and ODI, whereas the influences of housing type and temperature were subtle. Indoor site values were significantly positively correlated to outdoor sites' values for both OI and ODI. OI and ODI values from households were best predicted with those from public places at 1- and 0-week lags, respectively. Weekly rainfall values at 4- and 3-week lags were the best predictors of OI and ODI for households and public places, respectively. Monthly mean PHI, OI, and ODI were significantly associated with local dengue cases. In conclusion, ovitrap may be an effective tool for monitoring the population dynamics of Aedes mosquitoes, predicting dengue outbreaks, and serving as an early indicator to initiate environmental clean-up. Ovitrap surveillance is easy for surveyors if they are tasked with a certain number of ovitraps at a designated area, unlike the existing larval surveillance methodology, which entails identifying potential breeding sites largely at the surveyors' discretion. Ovitrap surveillance may reduce the influence of individual effort in larval surveillance that likely causes inconsistency in results.

Generation of Variability in Chrysodeixis includens Nucleopolyhedrovirus (ChinNPV): The Role of a Single Variant.

The mechanisms generating variability in viruses are diverse. Variability allows baculoviruses to evolve with their host and with changes in their environment. We examined the role of one genetic variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and its contribution to the variability of the virus under laboratory conditions. A mixture of natural isolates (ChinNPV-Mex1) contained two genetic variants that dominated over other variants in individual larvae that consumed high (ChinNPV-K) and low (ChinNPV-E) concentrations of inoculum. Studies on the ChinNPV-K variant indicated that it was capable of generating novel variation in a concentration-dependent manner. In cell culture, cells inoculated with high concentrations of ChinNPV-K produced OBs with the ChinNPV-K REN profile, whereas a high diversity of ChinNPV variants was recovered following plaque purification of low concentrations of ChinNPV-K virion inoculum. Interestingly, the ChinNPV-K variant could not be recovered from plaques derived from low concentration inocula originating from budded virions or occlusion-derived virions of ChinNPV-K. Genome sequencing revealed marked differences between ChinNPV-K and ChinNPV-E, with high variation in the ChinNPV-K genome, mostly due to single nucleotide polymorphisms. We conclude that ChinNPV-K is an unstable genetic variant that is responsible for generating much of the detected variability in the natural ChinNPV isolates used in this study.

Cross-Resistance of the Codling Moth against Different Isolates of Cydia pomonella Granulovirus Is Caused by Two Different but Genetically Linked Resistance Mechanisms.

Cydia pomonella granulovirus (CpGV) is a widely used biological control agent of the codling moth. Recently, however, the codling moth has developed different types of field resistance against CpGV isolates. Whereas type I resistance is Z chromosomal inherited and targeted at the viral gene pe38 of isolate CpGV-M, type II resistance is autosomal inherited and targeted against isolates CpGV-M and CpGV-S. Here, we report that mixtures of CpGV-M and CpGV-S fail to break type II resistance and is expressed at all larval stages. Budded virus (BV) injection experiments circumventing initial midgut infection provided evidence that resistance against CpGV-S is midgut-related, though fluorescence dequenching assay using rhodamine-18 labeled occlusion derived viruses (ODV) could not fully elucidate whether the receptor binding or an intracellular midgut factor is involved. From our peroral and intra-hemocoel infection experiments, we conclude that two different (but genetically linked) resistance mechanisms are responsible for type II resistance in the codling moth: resistance against CpGV-M is systemic whereas a second and/or additional resistance mechanism against CpGV-S is located in the midgut of CpR5M larvae.

A Multidisciplinary Investigation of the First Chikungunya Virus Outbreak in Matadi in the Democratic Republic of the Congo.

Early March 2019, health authorities of Matadi in the Democratic Republic of the Congo alerted a sudden increase in acute fever/arthralgia cases, prompting an outbreak investigation. We collected surveillance data, clinical data, and laboratory specimens from clinical suspects (for CHIKV-PCR/ELISA, malaria RDT), semi-structured interviews with patients/caregivers about perceptions and health seeking behavior, and mosquito sampling (adult/larvae) for CHIKV-PCR and estimation of infestation levels. The investigations confirmed a large CHIKV outbreak that lasted February-June 2019. The total caseload remained unknown due to a lack of systematic surveillance, but one of the two health zones of Matadi notified 2686 suspects. Of the clinical suspects we investigated (n = 220), 83.2% were CHIKV-PCR or IgM positive (acute infection). One patient had an isolated IgG-positive result (while PCR/IgM negative), suggestive of past infection. In total, 15% had acute CHIKV and malaria. Most adult mosquitoes and larvae (>95%) were Aedes albopictus. High infestation levels were noted. CHIKV was detected in 6/11 adult mosquito pools, and in 2/15 of the larvae pools. This latter and the fact that 2/6 of the CHIKV-positive adult pools contained only males suggests transovarial transmission. Interviews revealed that healthcare seeking shifted quickly toward the informal sector and self-medication. Caregivers reported difficulties to differentiate CHIKV, malaria, and other infectious diseases resulting in polypharmacy and high out-of-pocket expenditure. We confirmed a first major CHIKV outbreak in Matadi, with main vector Aedes albopictus. The health sector was ill-prepared for the information, surveillance, and treatment needs for such an explosive outbreak in a CHIKV-naïve population. Better surveillance systems (national level/sentinel sites) and point-of-care diagnostics for arboviruses are needed.