Impact of Impaired Cholesterol Homeostasis on Neutrophils in Atherosclerosis.

Atherosclerosis is complex chronic disease characterized by intimal cholesterol accumulation and vascular inflammation. There is a well-established relationship of hypercholesterolemia and inflammation with atherosclerosis. However, the link between inflammation and cholesterol is not completely understood. Myeloid cells, in particular, monocytes, macrophages, and neutrophils play essential roles in the pathogenesis of atherosclerotic cardiovascular disease. It is well known that macrophages accumulate cholesterol, forming foam cells, which drive atherosclerosis-associated inflammation. However, the interaction between cholesterol and neutrophils remains poorly defined-an important gap in the literature given that neutrophils represent up to 70% of total circulating leukocytes in humans. Elevated levels of biomarkers of neutrophil activation (myeloperoxidase and neutrophil extracellular traps) and higher absolute neutrophil counts are both associated with increased rates of cardiovascular events. Neutrophils contain the necessary machinery to uptake, synthesize, efflux and esterify cholesterol; yet, the functional consequence of dysregulated cholesterol homeostasis on neutrophil activity remains poorly defined. Preclinical animal data suggest a direct link between cholesterol metabolism and hematopoiesis, although current evidence in humans has been unable to corroborate such findings. This review will explore the impact of impaired cholesterol homeostasis neutrophils and draw focus on the discordant data from animal models and atherosclerotic disease in humans.

Fibroblast growth factor 21 in heart failure.

Fibroblast growth factor 21 (FGF21) is a peptide hormone involved in energy homeostasis that protects against the development of obesity and diabetes in animal models. Its level is elevated in atherosclerotic cardiovascular diseases (CVD) in humans. However, little is known about the role of FGF21 in heart failure (HF). HF is a major global health problem with a prevalence that is predicted to rise, especially in ageing populations. Despite improved therapies, mortality due to HF remains high, and given its insidious onset, prediction of its development is challenging for physicians. The emergence of cardiac biomarkers to improve prediction, diagnosis, and prognosis of HF has received much attention over the past decade. Recent studies have suggested FGF21 is a promising biomarker candidate for HF. Preclinical research has shown that FGF21 is involved in the pathophysiology of HF through the prevention of oxidative stress, cardiac hypertrophy, and inflammation in cardiomyocytes. However, in the available clinical literature, FGF21 levels appear to be paradoxically raised in HF, potentially implying a FGF21 resistant state as occurs in obesity. Several potential confounding variables complicate the verdict on whether FGF21 is of clinical value as a biomarker. Further research is thus needed to evaluate whether FGF21 has a causal role in HF, and whether circulating FGF21 can be used as a biomarker to improve the prediction, diagnosis, and prognosis of HF. This review draws from preclinical and clinical studies to explore the role of FGF21 in HF.

An Evaluation of Characters for the Separation of Two Culex Species (Diptera: Culicidae) Based on Material From the Upper Midwest.

Mosquitoes (Diptera: Culicidae) in the Culex pipiens complex play a key role in the transmission and therefore epidemiology of a number of human and animal pathogens globally. These mosquitoes, and sympatric species of the genus Culex Linnaeus that are not within the Cx. pipiens complex, are often considered 'impossible' to distinguish by morphology in the adult female stage. In the United States, this is particularly true for Culex pipiens s.l. and Culex restuans Theobald, both of which are competent vectors of West Nile virus, but likely play different roles in the transmission cycle. Therefore, we undertook an in-depth morphological evaluation of matched larval exuviae and adult specimens that revealed five useful morphological characters that are informative to distinguish Cx. pipiens s.l. from Cx. restuans in the adult stage. Herein, we provide a comprehensive review of the literature on these species of interest, and four additional, morphologically similar, Culex species, and a proposed key to adult female specimens.

Average proportional consecutive interval difference accurately differentiates spontaneous activity from motor unit potentials.

INTRODUCTION: An objective method is required to detect spontaneous activity (SA) for prevalence studies in needle electromyography (EMG). Because of frequent similarities in the morphology of SA and motor unit potentials (MUP), identification of SA depends on assessment of firing regularity, which has not yet been quantitated through a modern interface. METHODS: Prospective recordings obtained from patients referred for electrodiagnostic evaluation were analyzed by using decomposition-based quantitative EMG (DQEMG) customized to calculate descriptive statistics. RESULTS: Forty-four MUP recordings (39 participants) and 80 SA recordings (62 participants) were analyzed. One hundred one of 124 recordings successfully interfaced with DQEMG. The remaining recordings were analyzed in Audacity. Average proportional consecutive interval differences differentiated SA from MUPs with 97.5% sensitivity (confidence interval [CI] 91.3%-99.7%) and 100.0% specificity (CI 92%-100%). There was substantial overlap, however, for SD and mean consecutive differences. DISCUSSION: Average proportional consecutive interval difference accurately differentiates SA from MUPs and may be useful in future prevalence studies of SA.

Colchicine in atherosclerotic cardiovascular disease.

Inflammation has a direct role in the development of atherosclerotic vascular disease, and oral colchicine displays broad anti-inflammatory properties. Several large, randomised controlled trials (RCTs) have evaluated colchicine's impact on cardiovascular outcomes. Results from a meta-analysis of these trials demonstrate that colchicine reduces the risk of recurrent major adverse cardiovascular events (MACEs) by 25%, leading to its recent approval by the Food and Drug Administration for the treatment and prevention of cardiovascular disease. Despite this, colchicine has not been shown to confer any survival benefit in these trials. The non-significant reduction in cardiovascular death of 18% (95% CI: 45% decrease to 23% increase) is outweighed by a more prominent, borderline non-significant increase in the risk of non-cardiovascular death by 38% (95% CI: 1% decrease to 92% increase). Key populations including those with heart failure, those undergoing surgical revascularisation, women, elderly individuals and non-Caucasians are under-represented in completed trials, which limits generalisability. C reactive protein has been proposed as a biomarker for colchicine response and shows promise for identifying a high-risk population where the benefit on MACE reduction and specifically reduced cardiovascular death might outweigh any real increased risk of non-cardiovascular death; however, this approach is still to be validated in ongoing RCTs. In conclusion, while colchicine shows promise in reducing MACE, its net risk-benefit profile requires further elucidation before its widespread adoption into clinical practice for the secondary prevention of atherosclerotic cardiovascular disease. Much more large-scale, long-term trial data are still needed in this space.

Association of circulating fibroblast growth factor 21 levels with all-cause and cardiovascular mortality: The multi-ethnic study of atherosclerosis.

BACKGROUND: Fibroblast growth factor 21 (FGF21) levels are often elevated in cardiovascular disease (CVD). However, no study has assessed its association with cardiovascular and all-cause mortality in a population free of clinically evident CVD. METHODS: A total of 5543 Multi-Ethnic Study of Atherosclerosis (MESA) participants (mean age 62.7 years, 47.5 % male), free of clinically evident CVD at baseline, were studied. From baseline (2000-2002), 1606 deaths (including 387 CVD deaths) were observed over a median follow-up of 17.7 years. Multivariable Cox regression analysis was performed to assess the association of plasma FGF21 levels with mortality. RESULTS: FGF21 levels at baseline were associated with all-cause mortality, even after adjustment for traditional risk factors, including demographic, socioeconomic and cardiovascular risk factors (adjusted hazard ratio 1.08 [95% confidence interval 1.01, 1.16] per 1 SD increase in ln-transformed levels; 1.27 for the highest vs, lowest quartile). Baseline FGF21 levels were significantly associated with both CVD and non-CVD mortality in unadjusted models. However, the association with non-CVD mortality, but not CVD mortality, remained statistically significant after adjusting for covariates. Similar results were obtained in FGF21 quartile analyses and also when using competing risk regression or matched case-control cohort in sensitivity analyses. CONCLUSIONS: In subjects without clinically-evident CVD at baseline, over 17.7 years follow-up there is a modest association of baseline FGF21 levels with all-cause mortality. The finding that this is driven primarily by a significant association with non-CVD mortality over almost two decades merits further investigation.

Improvement in "Jumping Stump" Syndrome Following Diagnostic Sciatic Nerve Block and Home Exercise Program in a Transtibial Amputee.

"Jumping Stump" syndrome is a rare postoperative complication seen in the residual limb of amputees, with only a few cases documented in the literature. It has been defined as a peripherally induced movement disorder leading to either dystonia, myoclonus, tremors, or choreiform movements in the amputated residual limb. It is often associated with significant discomfort and an inability to ambulate with a prosthetic limb. Treatment options remain inconclusive at this time. We present a case of "Jumping Stump" syndrome in a young female transtibial amputee following revision transtibial amputation (TTA) with myodesis and targeted muscle reinnervation. About six weeks after revision surgery, the patient started experiencing significant myoclonus of the right residual limb when extending the knee. She was trialed on various oral pharmacologic agents over six months and had multiple prosthetic adjustments without any symptomatic relief. Moreover, the patient was also prescribed a daily knee range of motion (ROM) and stretching program. Six months after symptom onset, she underwent a diagnostic right sciatic nerve block and right biceps femoris point block with immediate and significant improvement in symptoms. She had a greater ROM in the affected limb without myoclonus and was able to ambulate once again with her prosthetic limb. Our patient's response to a diagnostic nerve and motor point block, as well as her marked improvement of symptoms with a consistent home exercise (stretching) program, suggests that desensitization of a muscle-tendon stretch response likely accounted for the improvement of symptoms. It is hypothesized that chemodenervation via botulinum toxin, in addition to the consistent home stretching program, would have accelerated the improvement of symptoms and should be further explored as a potential treatment modality for "Jumping Stump" syndrome.

Thrombolysis of bowel obstruction? A thought-provoking presentation of a common surgical pathology.

We present a unique case of bowel obstruction with a hiatus hernia causing atypical chest pain with dynamic ST-segment elevation in a regional Australian emergency department. The ST elevation only resolved after nasogastric decompression of the bowel obstruction. Early thrombolysis of presumed myocardial infarction led to upper gastrointestinal tract bleeding that could have been avoided with timely diagnosis. An extensive review of literature, in addition to our case report, suggests bowel obstruction is a differential diagnosis for patients who have inferior pattern ST elevation but normal troponin presenting with atypical chest pain, nausea, vomiting and previous abdominal surgery.

Acute Coronary Syndrome: Unravelling the Biology to Identify New Therapies.

Acute coronary syndrome (ACS) encompasses a spectrum of presentations including unstable angina, non-ST elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI) [...].

Outbreak Investigation: Jamestown Canyon Virus Surveillance in Field-Collected Mosquitoes (Diptera: Culicidae) From Wisconsin, USA, 2018-2019.

During the summers of 2017-2019, 60 human cases of Jamestown Canyon virus-associated disease were reported in the State of Wisconsin, U.S.A; by comparison, there were 28 cases in the 5 years prior. Jamestown Canyon virus (JCV, Peribunyaviridae: Orthobunyavirus) is a zoonotic, mosquito-borne virus that is endemic throughout North America. The proposed transmission cycle for JCV involves horizontal transmission by a variety of mammal-feeding mosquito species and deer hosts, and transseasonal maintenance by vertical transmission in Aedes mosquito species. Although some of the earliest work on JCV transmission and disease was done in Wisconsin (WI), little is known about the spectrum of mosquitoes that are currently involved in transmission and maintenance of JCV, which is key to inform the approach to control and prevent JCV transmission, and to understand why case numbers have increased dramatically in recent years. Therefore, we undertook an intensive surveillance effort in Sawyer and Washburn counties, WI between April and August of 2018 and 2019, in an area with a concentration of JCV human cases. Larval and adult stages of mosquitoes were surveyed using larval dippers and emergence traps, light traps, resting boxes, a Shannon-style trap, and backpack aspirator. In total, 14,949 mosquitoes were collected in 2018, and 28,056 in 2019; these specimens represent 26 species in 7 genera. Suspect vector species were tested for JCV by polymerase chain reaction (PCR); of 23 species that were tested, only Aedes provocans yielded JCV positive results. In 2018, a single pool of Ae. provocans tested positive. In 2019, with more focused early season surveillance, we detected JCV in 4 pools of adult mosquitoes, and one pool that consisted of lab-raised adults that were collected as larvae. Material from all of these PCR-positive samples also yielded infectious virus in cell culture. Overall, these data provide new insight into the seasonality and habitat preferences for 26 mosquito species in Northern WI, which will be useful to inform future surveillance efforts for JCV. The results underscore the importance of Ae. provocans as a vector species involved in transseasonal maintenance of JCV in this region.

Characterizing oogenesis and programmed cell death in the eastern tree hole mosquito Aedes (Protomacleaya) triseriatus.

Oogenesis in flies manifests as a carefully orchestrated cascade of developmental gates and growth events, punctuated by programmed cell death (PCD) and follicular resorption events. In anautogenous mosquitoes, a blood meal stimulates growth of primary follicles, but the timing of developmental stages is species-specific, and few species have been characterized. Here, we characterize the first gonotrophic cycle of oogenesis in Aedes triseriatus (Diptera: Culicidae), the principal vector of La Crosse Virus (LACV), a major cause of pediatric encephalitis in North America. We note significant differences in the timing and appearance of developmental stages from previous studies of other mosquito species, particularly Aedes aegypti. We also describe the appearance and timing of PCD events including atresia, nurse cell death, and follicular epithelium death and show that the majority of follicular epithelium cells do not undergo apoptosis during oogenesis but persist in the ovariole at least until the second gonotrophic cycle. This thorough characterization of oogenesis and PCD in Ae. triseriatus, through which LACV must persist in order to achieve filial infection, also serves as a baseline to study host-pathogen interactions during transovarial transmission.

Snow-Covered Tires Generate Microhabitats That Enhance Overwintering Survival of Aedes albopictus (Diptera: Culicidae) in the Midwest, USA.

The Asian tiger mosquito, Aedes albopictus (Skuse), is a public health threat because it can potentially transmit multiple pathogenic arboviruses, exhibits aggressive diurnal biting, and is highly invasive. As Ae. albopictus moved northward into the United States, the limits of expansion were predicted as locations with a mean January temperature warmer than -2.5°C. We postulated that the range of Ae. albopictus could exceed these temperature limits if eggs in diapause overwinter in tires that provide an insulating effect from extreme temperatures. Fifteen tires with Ae. albopictus and Aedes triseriatus (Say) eggs, a native cold hardy species, were placed outside at five locations along a latitudinal gradient in Wisconsin and Illinois during the winter of 2018-2019; notably, in January 2019, a regional arctic air event brought the lowest temperatures recorded in over 20 yr. External and internal tire temperatures were recorded at 3 hr intervals, and egg survival was recorded after six months. Aedes albopictus eggs survived only from tires at northernmost locations. The mean internal January temperature of tires that supported survival was -1.8°C, while externally the mean temperature was -5.3°C, indicating that tires provided an average of +3.5°C of insulation. Tires that supported egg survival also had over 100 mm of snow cover during January. In the absence of snow cover, tires across the study area provided an average +0.79°C [95% CI 0.34-1.11] insulation. This work provides strong argument for the inclusion of microhabitats in models of dispersal and establishment of Ae. albopictus and other vector species.

Acute Coronary Syndromes (ACS)-Unravelling Biology to Identify New Therapies-The Microcirculation as a Frontier for New Therapies in ACS.

In acute coronary syndrome (ACS) patients, restoring epicardial culprit vessel patency and flow with percutaneous coronary intervention or coronary artery bypass grafting has been the mainstay of treatment for decades. However, there is an emerging understanding of the crucial role of coronary microcirculation in predicting infarct burden and subsequent left ventricular remodelling, and the prognostic significance of coronary microvascular obstruction (MVO) in mortality and morbidity. This review will elucidate the multifaceted and interconnected pathophysiological processes which underpin MVO in ACS, and the various diagnostic modalities as well as challenges, with a particular focus on the invasive but specific and reproducible index of microcirculatory resistance (IMR). Unfortunately, a multitude of purported therapeutic strategies to address this unmet need in cardiovascular care, outlined in this review, have so far been disappointing with conflicting results and a lack of hard clinical end-point benefit. There are however a number of exciting and novel future prospects in this field that will be evaluated over the coming years in large adequately powered clinical trials, and this review will briefly appraise these.

Inflammation during Percutaneous Coronary Intervention-Prognostic Value, Mechanisms and Therapeutic Targets.

Periprocedural myocardial injury and myocardial infarction (MI) are not infrequent complications of percutaneous coronary intervention (PCI) and are associated with greater short- and long-term mortality. There is an abundance of preclinical and observational data demonstrating that high levels of pre-, intra- and post-procedural inflammation are associated with a higher incidence of periprocedural myonecrosis as well as future ischaemic events, heart failure hospitalisations and cardiac-related mortality. Beyond inflammation associated with the underlying coronary pathology, PCI itself elicits an acute inflammatory response. PCI-induced inflammation is driven by a combination of direct endothelial damage, liberation of intra-plaque proinflammatory debris and reperfusion injury. Therefore, anti-inflammatory medications, such as colchicine, may provide a novel means of improving PCI outcomes in both the short- and long-term. This review summarises periprocedural MI epidemiology and pathophysiology, evaluates the prognostic value of pre-, intra- and post-procedural inflammation, dissects the mechanisms involved in the acute inflammatory response to PCI and discusses the potential for periprocedural anti-inflammatory treatment.

The association of serum lipid and lipoprotein levels with total and differential leukocyte counts: Results of a cross-sectional and longitudinal analysis of the UK Biobank.

BACKGROUND AND AIMS: There is some evidence of a cross-sectional, and possibly causal, relationship of lipid levels with leukocyte counts in mice and humans. This study investigates the cross-sectional and longitudinal relationship of blood lipid and lipoprotein levels with leukocyte counts in the UK Biobank cohort. METHODS: The primary cross-sectional analysis included 417,132 participants with valid data on lipid measures and leukocyte counts. A subgroup analysis was performed in 333,668 participants with valid data on lipoprotein(a). The longitudinal analysis included 9058 participants with valid baseline and follow-up data on lipid and lipoprotein levels and leukocyte counts. The association of lipid and lipoprotein levels with leukocyte counts was analysed by multivariable linear regression. RESULTS: Several relationships were significant in both cross-sectional and longitudinal analysis. After adjustment for demographic, socioeconomic and other confounding factors, a higher eosinophil count was associated with lower HDL cholesterol and apolipoprotein A-I concentration (p < 0.001). Higher triglycerides levels were associated with higher total leukocyte, basophil, eosinophil, monocyte and neutrophil counts (all p < 0.01). A higher lymphocyte count was associated with a higher apolipoprotein B level (p < 0.001). In the longitudinal analysis, lipoprotein(a) was inversely associated with basophil count in men but not women (p < 0.001). CONCLUSIONS: Triglyceride levels demonstrate a robust positive association with total and differential leukocyte counts suggesting they may be directly involved in leukogenesis. However, unlike in murine models, the remainder of these relationships is modest, which suggests that cholesterol and lipoproteins are minimally involved in leukogenesis in humans.

The association between lipid levels and leukocyte count: A cross-sectional and longitudinal analysis of three large cohorts.

Background: Relationships between dyslipidaemia and leukocyte counts have been investigated in several studies, demonstrating limited evidence of associations in humans. As such, studying a diverse range of cohorts will ensure evidence is robust. This study focused on investigating cross-sectional and longitudinal relationships in three large-scale cohorts. Methods: The cross-sectional analysis included a total of 27,566 participants with valid data on lipid measures and leukocyte counts from three study cohorts: National Health and Nutrition Survey (NHANES), Korean National Health and Nutrition Survey (KNHANES) and Treating to New Targets (TNT) trial. The longitudinal analysis included 9323 participants with valid data on lipid measures and leukocyte counts at baseline and one year with statin treatment. Associations between lipid levels and leukocyte counts were analysed by multivariable linear regression and adjusted for basic demographic and cardiovascular risk factors. Results: Cross-sectional data from NHANES demonstrated the association of lower high-density lipoprotein (HDL) cholesterol and higher triglycerides with higher leukocyte count (0.9% lower and 0.3% higher count per 10 mg/dL increase in HDL cholesterol and triglycerides respectively, both p < 0.001). Similar trends were found in TNT trial (both p < 0.001), but not in KNHANES. In the TNT trial, 10 mg/dL increase in triglycerides over one year was also associated with a 0.09 × 103/µL increase in leukocyte count over the same period. Conclusions: The findings of this study are consistent with those of previous human studies, supporting weak yet noteworthy associations between dyslipidaemia and leukocytosis.

Colchicine Inhibits Neutrophil Extracellular Trap Formation in Patients With Acute Coronary Syndrome After Percutaneous Coronary Intervention.

Background Release of neutrophil extracellular traps (NETs) after percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS) is associated with periprocedural myocardial infarction, as a result of microvascular obstruction via pro-inflammatory and prothrombotic pathways. Colchicine is a well-established anti-inflammatory agent with growing evidence to support use in patients with coronary disease. However, its effects on post-PCI NET formation in ACS have not been explored. Methods and Results Sixty patients (40 ACS; 20 stable angina pectoris) were prospectively recruited and allocated to colchicine or no treatment. Within 24 hours of treatment, serial coronary sinus blood samples were collected during PCI. Isolated neutrophils from 10 patients with ACS post-PCI and 4 healthy controls were treated in vitro with colchicine (25 nmol/L) and stimulated with either ionomycin (5 µmol/L) or phorbol 12-myristate 13-acetate (50 nmol/L). Extracellular DNA was quantified using Sytox Green and fixed cells were stained with Hoechst 3342 and anti-alpha tubulin. Baseline characteristics were similar across both treatment and control arms. Patients with ACS had higher NET release versus patients with stable angina pectoris (P<0.001), which was reduced with colchicine treatment (area under the curve: 0.58 versus 4.29; P<0.001). In vitro, colchicine suppressed unstimulated (P<0.001), phorbol 12-myristate 13-acetate-induced (P=0.009) and ionomycin-induced (P=0.002) NET formation in neutrophils isolated from patients with ACS post-PCI, but not healthy controls. Tubulin organization was impaired in neutrophils from patients with ACS but was restored by colchicine treatment. Conclusions Colchicine suppresses NET formation in patients with ACS post-PCI by restoring cytoskeletal dynamics. These findings warrant further investigation in randomized trials powered for clinical end points. Registration URL: https://anzctr.org.au; Unique identifier: ACTRN12619001231134.

Relationship of fibroblast growth factor 21 levels with inflammation, lipoproteins and non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is associated with inflammation and atherogenic lipoprotein abnormalities. Previous studies suggest an association of fibroblast growth factor 21 (FGF21) with NAFLD. Therefore, we assessed the association of circulating FGF21 levels with inflammatory markers, lipoprotein profile and NAFLD in the Multi-Ethnic Study of Atherosclerosis (MESA). METHODS: Among 6814 participants free of apparent cardiovascular disease at baseline (2000-2002), 3634 participants had valid data on variables of interest. After excluding participants with excessive alcohol consumption, 3446 participants were included in the analysis. NAFLD was defined using non-contrast cardiac computed tomography with a liver-to-spleen ratio (LSR) < 1 or liver attenuation <40 Hounsfield units (HU). RESULTS: The mean age of the participants was 63.5 years with 54% females, 36% Caucasian, 10% Chinese American, 31% African American and 23% Hispanic. 17% of the participants had NAFLD. After adjustment for demographic, socioeconomic and other confounders, a 1-SD increment in ln-transformed FGF21 level was associated with a 5.1% higher IL-6 level, a 0.31 nm larger very-low-density lipoprotein particle diameter, a 0.014 nm smaller high-density lipoprotein particle diameter, and a 5.25 nmol/L lower intermediate-density lipoprotein particle concentration (all p < 0.05). A 1-SD increment in ln-transformed FGF21 level was associated with LSR<1 and liver attenuation <40 HU (OR = 1.38 and 1.48; both p < 0.01), even after adjusting for the aforementioned inflammation and lipoprotein parameters. CONCLUSIONS: This study suggests an association between FGF21 and NAFLD, independent of inflammation and atherogenic lipoprotein abnormalities. Further studies are needed to assess FGF21 as a biomarker for future NAFLD risk.

Arrival and establishment of Aedes japonicus japonicus (Diptera: Culicidae) in Iowa.

The arrival and establishment of Aedes (Finlaya) japonicus japonicus (Theobald) (Diptera: Culicidae) in Iowa are reported. In total, 518 wild adult specimens were collected through the statewide mosquito and mosquito-borne virus surveillance program in 2007 and 2008. Specimens were collected with New Jersey light traps, CO2-baited CDC light traps, grass infusion-baited gravid traps, and Mosquito Magnet traps located in 12 counties in central and eastern Iowa Specimens were identified morphologically, and identity was further supported by molecular DNA barcoding. Specimens also were tested for infection with West Nile virus (family Flaviviridae, genus Flavivirus, WNV) and La Crosse virus (family Bunyaviridae, genus Bunyavirus, LACV) by reverse transcription-polymerase chain reaction. Although no specimens tested positive for arbovirus infection, the arrival of Ae. j. japonicus in Iowa is a public health concern considering its potential to transmit several arboviruses, particularly WNV and LACV.

Genomic sequence and phylogenetic analysis of Culex flavivirus, an insect-specific flavivirus, isolated from Culex pipiens (Diptera: Culicidae) in Iowa.

Adult mosquitoes (Diptera: Culicidae) were collected in 2007 and tested for specific viruses, including West Nile virus, as part of the ongoing arbovirus surveillance efforts in the state of Iowa. A subset of these mosquitoes (6,061 individuals in 340 pools) was further tested by reverse transcription-polymerase chain reaction (RT-PCR) using flavivirus universal primers. Of the 211 pools of Culex pipiens (L.) tested, 50 were positive. One of 51 pools of Culex tarsalis Coquillet was also positive. The flavivirus minimum infection rates (expressed as the number of positive mosquito pools per 1,000 mosquitoes tested) for Cx. pipiens and Cx. tarsalis were 10.3 and 1.2, respectively. Flavivirus RNA was not detected in Aedes triseriatus (Say) (52 pools), Culex erraticus (Dyar & Knab) (25 pools), or Culex territans Walker (one pool). Sequence analysis of all RT-PCR products revealed that the mosquitoes had been infected with Culex flavivirus (CxFV), an insect-specific virus previously isolated in Japan, Indonesia, Texas, Mexico, Guatemala and Trinidad. The complete genome of one isolate was sequenced, as were the envelope protein genes of eight other isolates. Phylogenetic analysis revealed that CxFV isolates from the United States (Iowa and Texas) are more closely related to CxFV isolates from Asia than those from Mexico, Guatemala, and Trinidad.