Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Arboviruses can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important arboviruses, including dengue virus (DENV), Zika virus (ZIKV), Chikungunya (CHIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of DENV (as a model system for mosquito-borne viruses more generally) that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP)-the time taken for DENV virus to be transmissible after infection-is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicate that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection.

A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Systematic classification and comparison of maternal and obstetrical complications following 2 different methods of fetal surgery for the repair of open neural tube defects.

BACKGROUND: In utero repair of open neural tube defects using an open hysterotomy approach (hereafter referred to as "open") has been shown to reduce the need for ventriculoperitoneal shunting and to improve motor outcomes for affected infants. Laparotomy-assisted fetoscopic repair (hereafter referred to as "hybrid") is an alternative approach that may confer similar neurologic benefits while reducing the incidence of hysterotomy-related complications. OBJECTIVE: This study aimed to analyze procedure-related maternal and fetal complications of in utero repair using the Clavien-Dindo classification, and to compare the outcomes of the hybrid and open approaches. STUDY DESIGN: This was a retrospective cohort study conducted in a single center between September 2011 and July 2021. All patients who met the Management of Myelomeningocele Study criteria and who underwent either hybrid or open fetal surgery were included. Maternal complications were classified using a unique adaptation of the Clavien-Dindo scoring system, allowing the development of a comprehensive complication index score specific to fetal surgery. Primary fetal outcome was defined as gestational age at delivery and summarized according to the World Health Organization definitions of preterm delivery. RESULTS: There were 146 fetuses with open neural tube defects who were eligible for, and underwent, in utero repair during the study period. Of these, 102 underwent hybrid fetoscopic repair and 44 underwent open hysterotomy repair. Gestational age at the time of surgery was higher in the hybrid group than in the open group (25.1 vs 24.8 weeks; P=.004). Maternal body mass index was lower in the hybrid than in the open group (25.4 vs 27.1 kg/m2; P=.02). The duration of hybrid fetoscopic surgery was significantly longer in the hybrid than in the open group (250 vs 164 minutes; P<.001). There was a significantly lower Clavien-Dindo Grade III complication rate (4.9% vs 43.2%; P<.001) and a significantly lower overall comprehensive maternal complication index (8.7 vs 22.6; P=.021) in the hybrid group than in the open group. Gestational age at delivery was significantly higher in the hybrid group than in the open group (38.1 vs 35.8 weeks; P<.001), and this finding persisted when gestational age at delivery was analyzed using the World Health Organization definitions of preterm delivery. CONCLUSION: Use of our adaptation of the standardized Clavien-Dindo classification to assess the maternal complications associated with in utero open neural tube defect repair provides a new method for objectively assessing different fetal surgical approaches. It also provides a much-needed standardized tool to allow objective comparisons between methods, which can be used when counseling patients. The hybrid open neural tube defect repair was associated with lower rates of maternal adverse events , and later gestational age at delivery compared with the open approach.

Impact of the cystic neural tube defects on fetal motor function in prenatal myelomeningocele repairs: A retrospective cohort study.

OBJECTIVE: To determine the impact of the lesion type (cystic [myelomeningocele] or flat [myeloschisis]) on the fetal motor function (MF) in cases candidates for prenatal open neural tube defect (ONTD) repair. METHODS: Retrospective cohort study of patients with ONTD who underwent prenatal repair at a single institution between 2011 and 2019. The lesion type and the measurements of the length and width of the lesions to calculate the surface of the ellipsoid lesion were performed using MR scans. Prenatal MF of the lower extremities was evaluated by ultrasound following a metameric distribution at the time of referral. Intact MF was defined as the observation of plantar flexion of the ankle. Logistic regression was performed to determine the predictive value of the type of lesion for having an intact MF at the time of referral. RESULTS: 103 patients were included at 22.9 (19-25.4) weeks; 65% had cystic and 35% had flat lesions. At the time of referral, there was a higher proportion of cases with an intact MF in the presence of flat lesions (34/36; 94.4%) as compared to cystic lesion (48/67; 71.6%, p < 0.01). When adjusting for gestational age and anatomical level of the lesion, flat ONTD were 3.1 times more likely to be associated by intact motor function (CI%95 [2.1-4.6], p < 0.01) at the time of referral. CONCLUSION: Cystic ONTD are more likely to be associated with impaired MF at mid-gestation in candidates for prenatal ONTD repair.

Intertwin differences in umbilical artery pulsatility index are associated with infant survival in twin-to-twin transfusion syndrome.

OBJECTIVES: To evaluate the association of intertwin differences in umbilical artery pulsatility index (DUAPI) and infant survival in twin-to-twin transfusion syndrome (TTTS). METHODS: Absolute DUAPI was calculated prior to laser surgery. Receiver-operating characteristics (ROC) curve analysis provided an intertwin DUAPI cutoff of 0.4 for the prediction of double twin survival to 30 days of life. Infant survival was compared between women with an intertwin DUAPI <0.4 and ≥0.4 in the whole cohort, in TTTS cases with Quintero stages I/II and in those with Quintero stages III/IV. Regression analyses were performed to evaluate the association of intertwin DUAPI <0.4 and infant survival adjusted for confounders. RESULTS: In total, 349 TTTS cases were included. Double twin survival to 30 days was observed in 67% (234/349) of cases. Significant differences in double twin survival was seen between intertwin DUAPI groups in the whole cohort (76.8 vs. 52.2%; p<0.001), in women with TTTS Quintero stage I or II (77.8 vs. 58.5%; p=0.015) as well as in women with TTTS Quintero stage III or IV (75 vs. 49.5%; p=0.001). Intertwin DUAPI <0.4 conferred a threefold increased chance for double twin survival. CONCLUSIONS: Small intertwin DUAPI is associated with increased double infant survival in early and advanced TTTS stages.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Mosquito immune cells enhance dengue and Zika virus dissemination in Aedes aegypti.

Mosquito-borne viruses cause more than 400 million annual infections and place over half of the world's population at risk. Despite this importance, the mechanisms by which arboviruses infect the mosquito host and disseminate to tissues required for transmission are not well understood. Here, we provide evidence that mosquito immune cells, known as hemocytes, play an integral role in the dissemination of dengue virus (DENV) and Zika virus (ZIKV) in the mosquito Aedes aegypti. We establish that phagocytic hemocytes are a focal point for virus infection and demonstrate that these immune cell populations facilitate virus dissemination to the ovaries and salivary glands. Additional transfer experiments confirm that virus-infected hemocytes confer a virus infection to non-infected mosquitoes more efficiently than free virus in acellular hemolymph, revealing that hemocytes are an important tropism to enhance virus dissemination in the mosquito host. These data support a "trojan horse" model of virus dissemination where infected hemocytes transport virus through the hemolymph to deliver virus to mosquito tissues required for transmission and parallels vertebrate systems where immune cell populations promote virus dissemination to secondary sites of infection. In summary, this study significantly advances our understanding of virus infection dynamics in mosquitoes and highlights conserved roles of immune cells in virus dissemination across vertebrate and invertebrate systems.

Rapid, high-capacity adsorption of iodine from aqueous environments with amide functionalized covalent organic frameworks.

The uses and production of radionuclides in nuclear energy production and medical therapy are becoming more significant in today's world. While these applications have many benefits, they can produce harmful pollutants, such as radioactive iodine, that need to be sequestered. Effective capture and storage of radioactive iodine waste remains a major challenge for nuclear energy generation and nuclear medicine. Here we report the highly efficient capture of iodine in a series of mesoporous, two-dimensional (2D) covalent organic frameworks, called COFamides, which contain amide sidechains in their pores. COFamides are capable of rapidly removing iodine from aqueous solution at concentrations as low as 50 ppm, with total capacities greater than 650 wt%. In order to explain the high affinity of the COFamide series for iodine and iodide species in water, we performed a computational analysis of the interactions between the COFamide framework and iodine guests. These studies suggest that the origin of the large iodine capacity in these materials can be explained by the presence of multiple, cooperative, non-covalent interactions between the framework and both iodine, and iodide species.

Hierarchically Structured Metal-Organic Framework Polymer Composites for Chemical Warfare Agent Degradation.

Metal-organic frameworks (MOFs) have captured the imagination of researchers for their highly tunable properties and many potential applications, including as catalysts for a variety of transformations. Even though MOFs possess significant potential, the challenges associated with processing of these crystalline powders into usable form factors while retaining their functional properties limit their end use applications. Herein, we introduce a new approach to construct MOF-polymer composites via 3D photoprinting to overcome these limitations. We designed photoresin composite formulations that use polymerization-induced phase separation to cause the MOF catalysts to migrate to the surface of the printed material, where they are accessible to substrates such as chemical warfare agents. Using our approach, MOF-polymer composites can be fabricated into nearly any shape or architecture while retaining both the excellent catalytic activity at 10 wt % loading of the MOF components and the flexible, elastomeric mechanical properties of a polymer.

Sindbis virus is suppressed in the yellow fever mosquito Aedes aegypti by ATG-6/Beclin-1 mediated activation of autophagy.

Autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains underexplored. To address this gap, we use a genetic approach to activate macroautophagy/autophagy in the yellow fever mosquito (Aedes aegypti), infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17-amino acid peptide derived from the Ae. aegypti autophagy-related protein 6 (ATG-6/beclin-1-like protein) is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of ATG-8 and puncta formation. Next, we engineered a recombinant SINV expressing this bioactive beclin-1-like peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulated production of infectious viruses. The results from this study improve our understanding of the role of autophagy in arboviruses in invertebrate hosts and also highlight the potential for the autophagy pathway to be exploited for arboviral control.

Increased blood meal size and feeding frequency compromise Aedes aegypti midgut integrity and enhance dengue virus dissemination.

Aedes aegypti is a highly efficient vector for numerous pathogenic arboviruses including dengue virus (DENV), Zika virus, and yellow fever virus. This efficiency can in part be attributed to their frequent feeding behavior. We previously found that acquisition of a second, full, non-infectious blood meal could accelerate virus dissemination within the mosquito by temporarily compromising midgut basal lamina integrity; however, in the wild, mosquitoes are often interrupted during feeding and only acquire partial or minimal blood meals. To explore the impact of this feeding behavior further, we examined the effects of partial blood feeding on DENV dissemination rates and midgut basal lamina damage in Ae. aegypti. DENV-infected mosquitoes given a secondary partial blood meal had intermediate rates of dissemination and midgut basal lamina damage compared to single-fed and fully double-fed counterparts. Subsequently, we evaluated if basal lamina damage accumulated across feeding episodes. Interestingly, within 24 hours of feeding, damage was proportional to the number of blood meals imbibed; however, this additive effect returned to baseline levels by 96 hours. These data reveal that midgut basal lamina damage and rates of dissemination are proportional to feeding frequency and size, and further demonstrate the impact that mosquito feeding behavior has on vector competence and arbovirus epidemiology. This work has strong implications for our understanding of virus transmission in the field and will be useful when designing laboratory experiments and creating more accurate models of virus spread and maintenance.

Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Flaviviruses are arthropod-borne (arbo)viruses which can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important flaviviruses, including dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of flaviviruses that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP) - the time taken for DENV virus to be transmissible after infection - is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicates that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection. Author summary: Aedes mosquitoes are the main vectors of dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), all of which can cause severe disease in humans with dengue alone infecting an estimated 100-400 million people each year. Understanding the processes that affect whether, and at which rate, mosquitoes may transmit such viruses is, hence, paramount. Here, we present a mathematical model of virus dynamics within infected mosquitoes. By combining the model with novel experimental data, we show that the course of infection is sensitive to the initial dose of virus ingested by the mosquito. The data also indicates that mosquitoes which blood feed subsequent to becoming infected may be able to transmit infection earlier, which is reproduced in the model. This is important as many mosquito species feed multiple times during their lifespan and, any reduction in time to dissemination will increase the number of days that a mosquito is infectious and so enhance the risk of transmission. Our study highlights the key and complementary roles played by mathematical models and experimental data for understanding within-mosquito virus dynamics.

Sexual transmission of Anopheles gambiae densovirus (AgDNV) leads to disseminated infection in mated females.

BACKGROUND: Anopheles gambiae densovirus (AgDNV) is an insect-specific, single-stranded DNA virus that infects An. gambiae sensu stricto (s.s.), the major mosquito species responsible for transmitting malaria parasites throughout sub-Saharan Africa. AgDNV is a benign virus that is very specific to its mosquito host and therefore has the potential to serve as a vector control tool via paratransgenesis (genetic modification of mosquito symbionts) to limit transmission of human pathogens. Prior to being engineered into a control tool, the natural transmission dynamics of AgDNV between An. gambiae mosquitoes needs to be fully understood. Additionally, improved knowledge of AgDNV infection in male mosquitoes is needed. In the study presented here, we examined the tissue tropism of AgDNV in the male reproductive tract and investigated both venereal and vertical transmission dynamics of the virus. METHODS: Anopheles gambiae s.s. adult males were infected with AgDNV via microinjection, and reproductive tissues were collected and assayed for AgDNV using qPCR. Next, uninfected females were introduced to AgDNV-infected or control males and, after several nights of mating, both the spermatheca and female carcass were assessed for venereally transmitted AgDNV. Finally, F1 offspring of this cross were collected and assayed to quantify vertical transmission of the virus. RESULTS: AgDNV infected the reproductive tract of male mosquitoes, including the testes and male accessory glands, without affecting mating rates. AgDNV-infected males venereally transmitted the virus to females, and these venereally infected females developed disseminated infection throughout the body. However, AgDNV was not vertically transmitted to the F1 offspring of this cross. CONCLUSIONS: Infected male releases could be an effective strategy to introduce AgDNV-based paratransgenic tools into naïve populations of An. gambiae s.s. females.

Balancing Self-Healing and Shape Stability in Dynamic Covalent Photoresins for Stereolithography 3D Printing.

Dynamic covalent bonds impart new properties to 3D printable materials that help to establish 3D printing as an accessible and efficient manufacturing technique. Here, we studied the effect of a thermally reversible Diels-Alder cross-linker on the shape stability of photoprintable resins and their self-healing properties. Resins containing different concentrations of dynamic covalent cross-links in a polyacrylate network showed that the content of dynamic cross-links plays a key role in balancing shape stability with self-healing ability. The shape stability of the printed objects was evaluated by measuring the dimensional changes after thermal treatment. The self-healing efficiency of the 3D printed resins was characterized with a scratch test and tensile testing. A dynamic covalent cross-link concentration of 1.8 mol % was enough to provide 99% self-healing efficiency without disrupting the shape stability of the printed objects. Our work shows the potential of dynamic covalent bonds in broadening the availability of 3D printable materials that are compatible with vat photopolymerization.

Impact of the volume of the myelomeningocele sac on imaging, prenatal neurosurgery and motor outcomes: a retrospective cohort study.

To investigate the association of the myelomeningocele (MMC) volume with prenatal and postnatal motor function (MF) in cases who underwent a prenatal repair. Retrospective cohort study (11/2011 to 03/2019) of 63 patients who underwent a prenatal MMC repair (37 fetoscopic, 26 open-hysterotomy). At referral, measurements of the volume of MMC was performed based on ultrasound scans. A large MMC was defined as greater than the optimal volume threshold (ROC analysis) for the prediction of intact MF at referral (2.7 cc). Prenatal or postnatal intact motor function (S1) was defined as the observation of plantar flexion of the ankle based on ultrasound scan or postnatal examination. 23/63 participants presented a large MMC. Large MMC lesions was associated with an increased risk of having clubfeet by 9.5 times (CI%95[2.1-41.8], p < 0.01), and reduces the chances of having an intact MF at referral by 0.19 times (CI%95[0.1-0.6], p < 0.01). At birth, a large MMC reduces the chance of having an intact MF by 0.09 times (CI%95[0.01-0.49], p < 0.01), and increases the risk of having clubfeet by 3.7 times (CI%95[0.8-18.3], p = 0.11). A lower proportion of intact MF and a higher proportion of clubfeet pre- or postnatally were observed in cases with a large MMC sac who underwent a prenatal repair.Trial registration: Clinicaltrials.gov NCT02230072 and NCT03794011 registered on September 3rd, 2014 and January 4th, 2019.

Maternal serum alpha-fetoprotein level and the relationship to ventriculomegaly in fetal neural tube defect: A retrospective cohort study.

OBJECTIVE: To estimate the significance of the association between mid-trimester maternal serum alpha-fetoprotein (MSAFP) level and fetal neuroanatomic findings in cases of open neural tube defect (ONTD). METHODS: Retrospective study of patients referred for prenatal ONTD repair between 2012 and 2018. Cases were classified into three groups based on their MSAFP level: 1)High MSAFP (>3.8MoM - n = 22), 2)Moderately high MSAFP (≤3.8 and ≥2.5MoM - n = 28), 3)Normal MSAFP (<2.5MoM - n = 18). MRI scans at the time of referral were used to assess the relationship between MSAFP and: A)Type of ONTD; B) Ventriculomegaly; C) Size of the myeloschisis lesion; D) Volume of myelomeningocele; E) Anatomical level of the lesion (LL). RESULTS: Having a high MSAFP level was more likely to be associated ventriculomegaly at mid-gestation than a moderately high or normal MSAFP level (OR = 8.4;CI95[0.9-73.4];p = 0.05 and OR = 2.8;CI95[0.9-8.8];p = 0.07). There were no differences between the three groups regarding type of lesion, size of the myeloschisis lesion, anatomic LL, or volume of the myelomeningocele sac. Myeloschisis cases with normal MSAFP had a larger surface area when compared to myeloschisis cases with moderately high MSAFP (219.8[104.4-551] vs 155.4[38.5-502.4] mm², p = 0.04). CONCLUSION: A 2nd trimester MSAFP level >3.8MoM in a fetus with ONTD is associated with mid-gestation ventriculomegaly.

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Anopheles gambiae densovirus (AgDNV) negatively affects Mayaro virus infection in Anopheles gambiae cells and mosquitoes.

BACKGROUND: Recent studies demonstrate that insect-specific viruses can influence the ability of their mosquito hosts to become infected with and transmit arboviruses of medical and veterinary importance. The aim of this study was to evaluate the interactions between Anopheles gambiae densovirus (AgDNV) (Parvoviridae) (a benign insect-specific virus that infects An. gambiae mosquitoes) and Mayaro virus (MAYV) (Togaviridae) (an emerging human pathogen that can be transmitted by An. gambiae) in both insect cell culture and mosquitoes. METHODS: For in vitro studies, An. gambiae Mos55 cells infected or uninfected with AgDNV were infected with MAYV. For in vivo studies, An. gambiae mosquitoes were injected intrathoracically with AgDNV and 4 days later orally infected with MAYV. Mosquitoes were dissected 10 days after MAYV infection, and MAYV titers in the body, legs and saliva samples quantified using focus-forming assay. RESULTS: MAYV virus replication was reduced 10-100-fold in An. gambiae Mos55 cells infected with AgDNV. In mosquitoes, there was a significant negative correlation between AgDNV and MAYV body titers 10 days post-blood meal. CONCLUSIONS: AgDNV infection was associated with reduced production of MAYV in cell culture, and reduced body titers of MAYV in An. gambiae mosquitoes. As densovirus infections are common in natural mosquito populations, these data suggest that they may affect the epidemiology of viruses of medical importance.