Genetic epidemiology of SARS-CoV-2 transmission in renal dialysis units - A high risk community-hospital interface.

OBJECTIVES: Patients requiring haemodialysis are at increased risk of serious illness with SARS-CoV-2 infection. To improve the understanding of transmission risks in six Scottish renal dialysis units, we utilised the rapid whole-genome sequencing data generated by the COG-UK consortium. METHODS: We combined geographical, temporal and genomic sequence data from the community and hospital to estimate the probability of infection originating from within the dialysis unit, the hospital or the community using Bayesian statistical modelling and compared these results to the details of epidemiological investigations. RESULTS: Of 671 patients, 60 (8.9%) became infected with SARS-CoV-2, of whom 16 (27%) died. Within-unit and community transmission were both evident and an instance of transmission from the wider hospital setting was also demonstrated. CONCLUSIONS: Near-real-time SARS-CoV-2 sequencing data can facilitate tailored infection prevention and control measures, which can be targeted at reducing risk in these settings.

Ascorbic acid lowers central blood pressure and asymmetric dimethylarginine in chronic kidney disease.

BACKGROUND: Premature cardiovascular disease in patients with chronic kidney disease (CKD) is not explained by traditional risk factors and oxidative stress may contribute via endothelial and vascular dysfunction. We investigated the effect of ascorbic acid on oxidative stress and vascular function in CKD patients compared with controls with hypertension (HTN). METHODS: A crossover study of intravenous saline and ascorbic acid was conducted. Biomarkers of oxidative stress were measured, while pulse wave analysis and brachial flow-mediated dilatation were performed to assess large artery and endothelial function. RESULTS: Twenty HTN and 30 CKD patients Stages 3-5 were recruited. Serum ascorbic acid was significantly lower in patients with CKD. In both groups, ascorbic acid significantly increased total antioxidant potential and superoxide. Asymmetric dimethylarginine (ADMA) was reduced significantly by ascorbic acid in the CKD group and on multivariate regression analysis, age and the presence of CKD were predictors of ADMA response to ascorbic acid. Although no effect on FMD was observed, central blood pressure and augmentation index were reduced significantly in both groups. CONCLUSIONS: Ascorbic acid has pro- and antioxidant effects, reducing central blood pressure and augmentation index in HTN and CKD. Ascorbic acid reduces serum ADMA in CKD, which may have longer-term benefits.

Non-Contrast Renal Magnetic Resonance Imaging to Assess Perfusion and Corticomedullary Differentiation in Health and Chronic Kidney Disease.

AIMS: Arterial spin labelling (ASL) MRI measures perfusion without administration of contrast agent. While ASL has been validated in animals and healthy volunteers (HVs), application to chronic kidney disease (CKD) has been limited. We investigated the utility of ASL MRI in patients with CKD. METHODS: We studied renal perfusion in 24 HVs and 17 patients with CKD (age 22-77 years, 40% male) using ASL MRI at 3.0T. Kidney function was determined using estimated glomerular filtration rate (eGFR). T1 relaxation time was measured using modified look-locker inversion and xFB02;ow-sensitive alternating inversion recovery true-fast imaging and steady precession was performed to measure cortical and whole kidney perfusion. RESULTS: T1 was higher in CKD within cortex and whole kidney, and there was association between T1 time and eGFR. No association was seen between kidney size and volume and either T1, or ASL perfusion. Perfusion was lower in CKD in cortex (136 ± 37 vs. 279 ± 69 ml/min/100 g; p < 0.001) and whole kidney (146 ± 24 vs. 221 ± 38 ml/min/100 g; p < 0.001). There was significant, negative, association between T1 longitudinal relaxation time and ASL perfusion in both the cortex (r = -0.75, p < 0.001) and whole kidney (r = -0.50, p < 0.001). There was correlation between eGFR and both cortical (r = 0.73, p < 0.01) and whole kidney (r = 0.69, p < 0.01) perfusion. CONCLUSIONS: Significant differences in renal structure and function were demonstrated using ASL MRI. T1 may be representative of structural changes associated with CKD; however, further investigation is required into the pathological correlates of reduced ASL perfusion and increased T1 time in CKD.

Inter-study reproducibility of arterial spin labelling magnetic resonance imaging for measurement of renal perfusion in healthy volunteers at 3 Tesla.

BACKGROUND: Measurement of renal perfusion is a crucial part of measuring kidney function. Arterial spin labelling magnetic resonance imaging (ASL MRI) is a non-invasive method of measuring renal perfusion using magnetised blood as endogenous contrast. We studied the reproducibility of ASL MRI in normal volunteers. METHODS: ASL MRI was performed in healthy volunteers on 2 occasions using a 3.0 Tesla MRI scanner with flow-sensitive alternating inversion recovery (FAIR) perfusion preparation with a steady state free precession (True-FISP) pulse sequence. Kidney volume was measured from the scanned images. Routine serum and urine biochemistry were measured prior to MRI scanning. RESULTS: 12 volunteers were recruited yielding 24 kidneys, with a mean participant age of 44.1 ± 14.6 years, blood pressure of 136/82 mmHg and chronic kidney disease epidemiology formula estimated glomerular filtration rate (CKD EPI eGFR) of 98.3 ± 15.1 ml/min/1.73 m2. Mean kidney volumes measured using the ellipsoid formula and voxel count method were 123.5 ± 25.5 cm3, and 156.7 ± 28.9 cm3 respectively. Mean kidney perfusion was 229 ± 41 ml/min/100 g and mean cortical perfusion was 327 ± 63 ml/min/100 g, with no significant differences between ASL MRIs. Mean absolute kidney perfusion calculated from kidney volume measured during the scan was 373 ± 71 ml/min. Bland Altman plots were constructed of the cortical and whole kidney perfusion measurements made at ASL MRIs 1 and 2. These showed good agreement between measurements, with a random distribution of means plotted against differences observed. The intra class correlation for cortical perfusion was 0.85, whilst the within subject coefficient of variance was 9.2%. The intra class correlation for whole kidney perfusion was 0.86, whilst the within subject coefficient of variance was 7.1%. CONCLUSIONS: ASL MRI at 3.0 Tesla provides a repeatable method of measuring renal perfusion in healthy subjects without the need for administration of exogenous compounds. We have established normal values for renal perfusion using ASL MRI in a cohort of healthy volunteers.

AA amyloidosis in a patient with Langerhans cell histiocytosis.

We report the case of a 37-year-old woman who presented with progressive renal dysfunction and proteinuria, in whom renal biopsy confirmed a diagnosis of AA amyloidosis. No evidence of chronic suppurative infection, connective tissue disease or malignancy was found. A past history of Langerhans cell histiocytosis (LCH) diagnosed in childhood was noted for which the patient had been successfully treated with surgical excision, corticosteroids, radiotherapy and chemotherapy. Renal disease in LCH is not widely recognized and thus we describe a patient with LCH in whom AA amyloidosis developed in the absence of any other established cause.

Simvastatin decreases lipopolysaccharide-induced pulmonary inflammation in healthy volunteers.

RATIONALE: Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option. OBJECTIVES: To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers. METHODS: Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n = 10/group) for 4 days before inhalation of 50 microg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) was measured in monocyte-derived macrophages. MEASUREMENTS AND MAIN RESULTS: Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-alpha, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P < 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-kappaB in monocyte-derived macrophages (P < 0.001); pretreatment with simvastatin reduced this by 35% (P < 0.001). CONCLUSIONS: Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS.

Influence of container size, location, and time of day on oviposition patterns of the dengue vector, Aedes aegypti, in Thailand.

We conducted a study to determine the effect of container size and location on oviposition site selection by Ae. aegypti in large outdoor field enclosures (10 x 10 x 4 m high). There was a strong positive relationship between increasing container diameter, container volume, and water surface area with egg numbers over both high (rainy, July) and low (cool-dry, January) dengue transmission seasons. Location of containers (indoors versus immediately outdoors and underneath houses) did not influence the number of eggs deposited for containers 5-32 cm in diameter in either season. No trends based on container color (black, brown, or grey) were observed. A slight trend with a greater numbers of eggs laid outdoors in the largest containers (42 cm diameter) during the dry season was observed. Three separate models were run using the mixed model procedure in SAS for each container attribute. Controlling for season, time, and date, the most important container attribute predicting total egg numbers was container volume (total capacity) explaining 88% of the variation, followed by water surface area (85%), and container diameter opening (83%). Oviposition peaked in the afternoon at 1600 hrs and 2000 hrs in the dry and rainy seasons, respectively. Few eggs were laid overnight (2000 hrs-0600 hrs). Our results indicate that physical attributes of oviposition sites, such as size, light-dark contrasts, and specular reflectance from water surfaces, play a significant role in oviposition site selection.

Standardizing container classification for immature Aedes aegypti surveillance in Kamphaeng Phet, Thailand.

For the development of community-based vector control programs for dengue prevention, one of the key components is to formulate an adequate classification scheme for the different containers in which immature Aedes mosquitoes develop. Such a standardized scheme would permit more efficient targeting of efforts and resources in the most productive way possible. Based on field data from Kamphaeng Phet, Thailand, we developed a classification method that consists of the shape (S), use (U), and material (M) of the container (SUM-method). We determined that by targeting the four container classes that held the most Ae. aegypti pupae, adult mosquito production could theoretically be reduced by 70%. The classification method may be equally suitable for similar studies elsewhere in the world. Main advantages of the classification scheme are that categorization of containers does not need to be done a priori, that there is no "miscellaneous" class, and that different immature control strategies can be easily and prospectively tested with a local database. We expect that the classification strategy will 1) facilitate comparison of results among different ecological and geographic settings and 2) simplify communication among vector control personnel and affected communities.

Spatial and temporal patterns in the recovery of Aedes aegypti (Diptera: Culicidae) populations after insecticide treatment.

Given that tools for dengue emergency control are limited, continuous evaluation of the effectiveness of insecticide applications in the field is of utmost importance. Such studies will provide a sound basis for defining spraying schemes for public health authorities in dengue-affected countries. In this article, we address the following research questions: How do different space spraying strategies affect Aedes aegypti (L.) (Diptera: Culicidae) populations in both space and time? More specifically, how well are these mosquitoes killed, and how quickly do their populations recover and from where? Field trials were carried out with ultralow volume sprayers in Kamphaeng Phet province, Thailand, with a pyrethrin mixture that was applied 1) indoors only, 2) indoors plus outdoors, 3) indoors with a doubled spraying time, and 4) indoors with doubled spraying time plus outdoors. We found that within 7 d, Ae. aegypti populations recovered to approximately 50% of their original numbers. Spraying the outdoor area and doubling the time sprayed per room only had a significant impact on mosquito numbers 1 d after spraying. Two and 7 d after spraying, these effects were no longer detected. By investigating the spatial arrangement of Ae. aegypti numbers, we found that during the first 2 d after spraying immigration from untreated areas extended approximately 15 m into the sprayed area, whereas after 7 d this effect extended up to 50 m. Results are discussed in relation to ongoing dengue control efforts in Thailand.

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Aedes aegypti (Diptera: Culicidae) production from non-residential sites in the Amazonian city of Iquitos, Peru.

Programmes for the surveillance of Aedes aegypti (L.) often focus on residential areas, ignoring non-residential sites. Between November 2003 and October 2004, pupal/demographic surveys were therefore conducted in non-residential sites in the Peruvian city of Iquitos. The sampled sites included schools, factories, ports, public markets, petrol stations, commercial zones, airports, government buildings, animal-production areas, and recreational areas. Compared with the residential sites that had been surveyed a few years earlier, the non-residential sites generally had fewer pupae/ha, even though pupae were found in a high percentage of the sites investigated. Nonetheless, although <56 pupae/ha were observed in the industrial, commercial, recreational and school sites, the river boats in the ports and the areas in and around public markets sometimes had pupal abundances (of 122-213 pupae/ha) that were comparable with those previously recorded in the residential sites. When the relative production of Ae. aegypti was calculated by container type and characteristic (lidded/lidless, indoors/outdoors, and water-use patterns), no single container category was found to be a major producer of Ae. aegypti, with the exception of flower vases in cemeteries. In general, almost all (97%) of the pupae collected in the non-residential sites came from unlidded containers, although 91% of those collected in river boats came from lidded storage areas. With the exception of lumber mills, plant nurseries and markets (where only 39%-60% of the pupae were collected outdoors), >70% of pupal production was outdoors. In commercial areas, 41% of the pupae came from manually-filled containers, compared with <12% in residential sites. These results indicate that non-residential sites can be highly productive for Ae. aegypti and that the role of such sites in dengue transmission requires further investigation.

The cytotoxic effects of human neutrophil peptide-1 (HNP1) and lactoferrin on oral squamous cell carcinoma (OSCC) in vitro.

Alpha-defensin or human neutrophil peptide-1 (HNP1) is a neutrophil-derived antimicrobial peptide with cytotoxic effects towards cancer cells. Lactoferrin is also stored in human neutrophils and is a glycoprotein involved in mediating cytotoxicity towards tumour cells. This study investigated the sensitivity of normal oral keratinocyte and oral squamous cell carcinoma (OSCC) cells to HNP1 and lactoferrin in various combinations. A concentration of 100 microg/ml HNP1 induced the most significant cytotoxic effect on both normal and OSCC cells. Lactoferrin (12.5, 25 and 250 microg/ml) also significantly induced cell death in OSCC cells after 72 h. Of note, a combination of 10 microg/ml HNP1 and 50 microg/ml lactoferrin induced a differential effect, not observed with either concentration alone, which stimulated proliferation in normal cells, but induced cell death in OSCC cells throughout the study. These results indicate a potentially important co-operative role for HNP1 and lactoferrin in facilitating a selective cytotoxic effect on tumour cells.

Evaluation of a sampling methodology for rapid assessment of Aedes aegypti infestation levels in Iquitos, Peru.

An epidemic of dengue during 2001 in Northwestern Peru reemphasized the need for efficient, accurate, and economical vector surveillance. Between November 1998 and January 1999, we carried out extensive entomological surveys in two neighborhoods of approximately 600 contiguous houses located in the Amazonian city of Iquitos, providing a unique opportunity to evaluate the Aedes aegypti (L.) rapid assessment survey strategy. Based on Pan American Health Organization recommendations, this strategy is used by the Peruvian Ministry of Health (MOH). In our analysis all household locations, including closed and unoccupied houses, were georeferenced and displayed in a geographic information system, which facilitated simulations of MOH surveys based on hypothetical systematic sampling transects. Larval, pupal, and adult mosquito indices were calculated for each simulation (n = 10) and compared with the indices calculated from the complete data set (n = 4). The range of indices calculated from simulations was moderately high, but included actual indices. For example, simulation ranges for house indices (HI, percentage of infested houses from complete survey) were 38-56% (45%), 36-42% (38%), 21-34% (30%), and 13-33% (27%) in four surveys. HI, Breteau index, pupae per hectare, adult index, and adults per hectare were more robust entomological indicators (coefficient of variation [CV]/mean = 0.1-2.9) than the container index, pupae per person, pupae per house, adults per person, and adults per house (CV/mean >20). Our results demonstrate that the MOH's Ae. aegypti risk assessment program provides reasonable estimates of indices based on samples from every house. However, it is critical that future studies investigate the association of these indices with rates of virus transmission to determine whether sampling variability will negatively impact the application of indices in a public health context.

Field validation of Aedes aegypti (Diptera: Culicidae) age estimation by analysis of cuticular hydrocarbons.

In previous studies, we developed linear regression models to age-grade female Aedes aegypti L. reared and maintained under controlled laboratory conditions. The models were based on temporal differences between two cuticular hydrocarbons, pentacosane (C25H52) and nonacosane (C29H60), which were extracted from Ae. aegypti legs and analyzed by gas-liquid chromatography. These initial models predicted adult female age up to 165 DD (12-15 calendar d at 28 degrees C). The age of older mosquitoes, however, could not be accurately predicted. In this study, our original regression models were tested using age data obtained from mosquitoes maintained in a field laboratory and those that were marked, released, and recaptured in northwestern Thailand. Our field data led to the development of two new regression models: one for the cool-dry season (February-March) and one for the rainy season (July-August). Both models resulted in better estimates of age than the original model and thus improved our ability to predict the age of Ae. aegypti to 15 calendar d. Females older than 15 d can be identified as such, but their exact age cannot yet be estimated. The new models will be useful for epidemiological studies and evaluating the impact of Ae. aegypti control interventions for disease prevention.

Comparison of dry ice-baited centers for disease control and New Jersey light traps for measuring mosquito abundance in California.

Mosquito catch in New Jersy light traps (NJLTs) has been declining in recent years, compromising the sensitivity of the California mosquito monitoring program. Centers for Disease Control traps (CDCTs) operated without light and augmented with dry ice have been considered for replacement or augmentation. To provide information on comparative sensitivity and ability to measure abundance over time and space, catch of mosquitoes in NJLTs was compared to catch in CDCTs operated concurrently at 8-10 sites within the Coachella Valley, Kern, San Joaquin County, and Sacramento-Yolo Mosquito and Vector Control Districts. The CDCTs always collected more female mosquitoes than did NJLTs; however, differences in sensitivity varied markedly over time and space precluding the calculation of a universal conversion factor. Regressions of the catch of female Culex tarsalis in CDCTs as a function of catch in NJLTs within districts indicated that the slopes varied markedly, again precluding the derivation of a universal function. Therefore, we recommend that mosquito surveillance programs replace or supplement NJLTs with systematically operated CDCTs to enhance sampling sensitivity for females of most mosquito species. However, both trap types should be operated concurrently at several sites within each district to derive regression functions to convert historical relative abundance data from NJLTs to equivalent counts in CDCTs for retrospective analyses.

The influence of pre-operative electrocardiographic abnormalities and cardiovascular risk factors on patient and graft survival following renal transplantation.

Premature cardiovascular disease (CVD) is the leading cause of mortality and of graft loss in renal transplant recipients. However, the pattern of cardiovascular risk factors (specifically modifiable risk factors) is not well established and may be different from the general population. In this study we investigated the importance of electrocardiographic abnormalities and conventional cardiovascular risk factors present at the time of first renal transplantation in a longitudinal follow-up study of 515 patients. Overall, 45.8% were cigarette smokers, 13.0% were diabetic, 75.1% had "hypertension", 12.2% had symptoms of angina pectoris and 9.1% had a past history of myocardial infarction or stroke. Two thirds of ECG tracings were abnormal. 58.7% of men and 37.5% of women had left ventricular hypertrophy (LVH). Overall, 28.2% had simple LVH, 20.5% had LVH with repolarisation changes ('strain'). 434 patients had complete data for multivariate analyses of patient and graft survival. A Cox multivariate analysis of patient survival (patients whose graft failed were censored in the analysis) identified: age (hazard ratio 1.03/year), diabetes (2.72), smoking (1.81) and family history of premature CVD (2.17) as independent risk factors for patient survival. An abnormal ECG was also independently associated with outcome, with the exception of isolated left ventricular hypertrophy. Left ventricular hypertrophy with strain, or ischaemic changes were associated with adverse outcome with a hazard ratio of 1.96 and 3.30 respectively. A similar analysis of the determinants of graft survival (patients who died with a functioning graft were censored in the analysis) identified: acute rejection (hazard ratio 2.38), cigarette smoking (1.48) and age (1.04/year) as independent predictors of graft failure. These data demonstrate a high prevalence of ECG abnormalities and CV risk factors in renal transplant recipients. Moreover, ECG abnormalities and "conventional" cardiovascular risk factors are associated with poor graft and patient outcome and represent potentially remediable risk factors for renal transplant recipients.

Cost effectiveness of three arbovirus surveillance methods in northern California.

We compared the cost effectiveness of enzootic arbovirus surveillance in northern California by antibody detection in sentinel chickens, virus isolation from mosquitoes, and antibody detection in wild avian hosts. Total and annual recurring costs were determined for each method based on estimated personnel and actual material and travel costs for biweekly surveillance at 3 sites in the Sacramento Valley from May 1 through mid-October 1997 and 1998. Serologic detection of antibodies in wild birds was the most expensive method. Total costs associated with sentinel chickens and mosquitoes combined were less than half of those for the wild bird program. Recurring annual costs for the wild bird and mosquito methods were only slightly less than expenses for those methods during the 1st year of operation, which included nonrecurring setup costs. Recurring costs for sentinel chickens were reduced approximately 40% from total costs during the 1st year of the program and were <14% of recurring costs for wild bird serology. Exceptions and caveats of our analysis are discussed. When considering data from a companion paper on detection of enzootic virus transmission using the 3 methods, we concluded that the current system that combines sentinel chickens and virus isolation from mosquitoes is the most cost-effective and efficient surveillance program and should be retained. Future research efforts should investigate the costs and surveillance efficiency of modifications in the frequency of specimen collection and the placement of chicken flocks and mosquito traps.

Analysis of survival of young and old Aedes aegypti (Diptera: Culicidac) from Puerto Rico and Thailand.

It generally is assumed that the daily probability of survival of wild adult mosquitoes is independent of age. To test this assumption we conducted mark-release-recapture studies in Puerto Rico and Thailand to determine if estimated daily survival rates between two different age cohorts of the dengue vector Aedes aegypti (L.) were the same. Survivorship was estimated with nonlinear regression analysis using bootstrapping to obtain estimates of errors. Initial recapture success of the younger cohort was greater than the older cohort at both locations. Our analysis revealed a significantly greater survival rate for the younger cohort of females in Puerto Rico, and no significant differences between age cohorts in Thailand. For comparison, a traditional approach for analyzing these type of data, linear regression of log-transformed captures over time (exponential model), was used to calculate the probability of daily survival based on slopes of linear regression lines for recaptured mosquitoes. With this method, the estimated daily survival rate of older females (13-23 d old) was significantly greater than survival of younger ones (3-13 d old) in Puerto Rico and Thailand. In addition, short-range movement of mosquitoes was observed in Puerto Rico; maximum dispersal distance detected was 79 m. Survival rates of adult Ae. aegypti may be age-dependent and nonlinear regression analysis is a sensitive approach for comparing patterns of mosquito survival based on mark, single release, multiple recapture data.

Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood?

Adult female Aedes aegypti (L.), the vector of dengue and yellow fever viruses, have an affinity for feeding on human blood and a tendency to forego feeding on sugar. This observation challenges two tenets of mosquito biology: (1) mosquitoes imbibe plant carbohydrates for synthesis of energy reserves and blood for reproduction and (2) egg production is reduced when mosquitoes feed on human blood compared with blood from other species. Sub-optimal amounts of the amino acid isoleucine in human blood (particularly free isoleucine in plasma) are thought to be responsible for lowered egg production when human blood is ingested. We tested the hypothesis that feeding on human blood is associated with a selective advantage for Ae. aegypti and is an underlying reason for this mosquito's intimate and epidemiologically important relationship with human beings. Our five experiments examined the effects of different isoleucine concentrations on accumulated energy reserves, frequency of host contact, survival, and egg production. When mosquitoes imbibed blood meals over a 7- to 10-d period and were not fed sugar, increased isoleucine concentration decreased energy reserves and did not increase egg production. Aedes aegypti took smaller but more frequent blood meals when feeding on a low-isoleucine human host daily compared with a high-isoleucine mouse host. Previous reports that isoleucine enhances egg production were confirmed only when females were fed sugar, an unusual behavior for most domestic Ae. aegypti populations. Females fed human blood and water had greater age-specific survival (l(x)), reproductive output (m(x)), and cumulative net replacement (R0) than cohorts fed human blood plus sugar or isoleucine-rich mouse blood with or without access to sugar. The unique isoleucine concentration of human blood is associated with Ae. aegypti's unusual propensity to feed preferentially and frequently on humans--a behavior that increases this mosquito's fitness, synthesis of energy reserves, and contact with human hosts, making it an especially effective disseminator of human pathogens.

Differential evolution of eastern equine encephalitis virus populations in response to host cell type.

Arthropod-borne viruses (arboviruses) cycle between hosts in two widely separated taxonomic groups, vertebrate amplifying hosts and invertebrate vectors, both of which may separately or in concert shape the course of arbovirus evolution. To elucidate the selective pressures associated with virus replication within each portion of this two-host life cycle, the effects of host type on the growth characteristics of the New World alphavirus, eastern equine encephalitis (EEE) virus, were investigated. Multiple lineages of an ancestral EEE virus stock were repeatedly transferred through either mosquito or avian cells or in alternating passages between these two cell types. When assayed in both cell types, derived single host lineages exhibited significant differences in infectivity, growth pattern, plaque morphology, and total virus yield, demonstrating that this virus is capable of host-specific evolution. Virus lineages grown in alternation between the two cell types expressed intermediate phenotypes consistent with dual adaptation to both cellular environments. Both insect-adapted and alternated lineages greatly increased in their ability to infect insect cells. These results indicate that different selective pressures exist for virus replication within each portion of the two-host life cycle, and that alternation of hosts selects for virus populations well adapted for replication in both host systems.