Repression of tick microRNA-133 induces organic anion transporting polypeptide expression critical for Anaplasma phagocytophilum survival in the vector and transmission to the vertebrate host.

The microRNAs (miRNAs) are important regulators of gene expression. In this study, we provide evidence for the first time to show that rickettsial pathogen Anaplasma phagocytophilum infection results in the down-regulation of tick microRNA-133 (miR-133), to induce Ixodes scapularis organic anion transporting polypeptide (isoatp4056) gene expression critical for this bacterial survival in the vector and for its transmission to the vertebrate host. Transfection studies with recombinant constructs containing transcriptional fusions confirmed binding of miR-133 to isoatp4056 mRNA. Treatment with miR-133 inhibitor resulted in increased bacterial burden and isoatp4056 expression in ticks and tick cells. In contrast, treatment with miR-133 mimic or pre-mir-133 resulted in dramatic reduction in isoatp4056 expression and bacterial burden in ticks and tick cells. Moreover, treatment of ticks with pre-mir-133 affected vector-mediated A. phagocytophilum infection of murine host. These results provide novel insights to understand impact of modulation of tick miRNAs on pathogen colonization in the vector and their transmission to infect the vertebrate host.

Characterization of tick organic anion transporting polypeptides (OATPs) upon bacterial and viral infections.

BACKGROUND: Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV). RESULTS: Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites. CONCLUSIONS: This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes.

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100µM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)2]Cl2, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)3]Cl3, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl2·2H2O) or cobalt(II) chloride hexahydrate (CoCl2·6H2O) alone had no effects as "free" cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. IMPORTANCE: Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly.

Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks.

The black-legged tick Ixodes scapularis transmits the human anaplasmosis agent, Anaplasma phagocytophilum. In this study, we show that A. phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), for its survival in ticks. RNAi analysis revealed that knockdown of isoatp4056 expression had no effect on A. phagocytophilum acquisition from the murine host but affected the bacterial survival in tick cells. Knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA significantly affected A. phagocytophilum survival and isoatp4056 expression in tick cells. Exogenous addition of XA induces isoatp4056 expression and A. phagocytophilum burden in both tick salivary glands and tick cells. Electrophoretic mobility shift assays provide further evidence that A. phagocytophilum and XA influences isoatp4056 expression. Collectively, this study provides important novel information in understanding the interplay between molecular pathways manipulated by a rickettsial pathogen to survive in its arthropod vector.

Concurrent delivery of tocotrienols and simvastatin by lipid nanoemulsions potentiates their antitumor activity against human mammary adenocarcenoma cells.

Tocotrienol rich fraction (TRF) of vitamin E was previously shown to have anticancer activity against murine tumor cells in vitro. TRF was also shown to potentiate the anticancer activity of statins. The objectives of this study were therefore (a) to prepare and characterize stable parenteral lipid nanoemulsions as a novel platform for the concurrent delivery of TRF and simvastatin for subsequent use in combination chemotherapy, and (b) to evaluate the antiproliferative activity of the nanoemulsions against MCF-7 and MDA-MB-231 human mammary tumor cells. Nanoemulsions were prepared by the high-pressure homogenization technique using a viscous 70/30 blend of TRF and medium chain triglycerides as the oil phase in which simvastatin was dissolved at 9%w/w loading. Nanoemulsion droplets were about 200 nm in size and had surface potential of -45 mV. In a dissolution study, approximately 20% of simvastatin was released in sink conditions after 24h. The stability of the nanoemulsions was monitored over 6 months of storage. No oxidation or degradation products were detected and no loss in simvastatin loading was observed during this period. The antiproliferative activity of the nanoemulsions was also retained after storage. The IC50 of the TRF nanoemulsions against MCF-7 and MDA-MB-231 was 14 and 7 µM, respectively, which decreased to 10 µM and 4.8 µM when simvastatin was added to the nanoemulsions. Nanoemulsions prepared with tocopherol had no anticancer activity and were used as negative control. This study demonstrated that parenteral lipid nanoemulsions are viable delivery platform for potential use in cancer chemotherapy.

Stability and in vitro antiproliferative activity of bioactive "Vitamin E" fortified parenteral lipid emulsions.

The objectives of this work were to engineer physically stable "Vitamin E" rich intravenous lipid emulsions and to evaluate their in vitro antiproliferative activity against MCF-7 (human mammary adenocarcinoma) and SW-620 (human colon adenocarcinoma) cell lines. Emulsions loaded with 70% vitamin E by total weight of the oil phase were stabilized with secondary emulsifiers and tested for their hemolytic effect and their plasma and electrolyte stability. Emulsions stabilized with sodium oleate and sodium deoxycholate were sensitive to electrolytes and exhibited significant hemolytic effect. On the other hand, addition of 2.5% poloxamer was found to stabilize the emulsions against electrolytes and physical stress, which was attributed to the steric effect of their polyoxyethylene (POE) head group. When tested for their antiproliferative effects, poloxamer-stabilized tocotrienol lipid emulsions were found to exhibit significantly higher anticancer activity than lipid emulsions enriched with tocopherol alone. The half maximal inhibitory concentrations (IC(50)) of tocotrienols lipid emulsions against MCF-7 and SW-620 were 14 and 12 µM, respectively, whereas the IC(50s) of tocopherol lipid emulsions were approximately 69 and 78 µM against MCF-7 and SW-620 cells, respectively.

Silent substitutions predictably alter translation elongation rates and protein folding efficiencies.

Genetic code redundancy allows most amino acids to be encoded by multiple codons that are non-randomly distributed along coding sequences. An accepted theory explaining the biological significance of such non-uniform codon selection is that codons are translated at different speeds. Thus, varying codon placement along a message may confer variable rates of polypeptide emergence from the ribosome, which may influence the capacity to fold toward the native state. Previous studies report conflicting results regarding whether certain codons correlate with particular structural or folding properties of the encoded protein. This is partly due to different criteria traditionally utilized for predicting translation speeds of codons, including their usage frequencies and the concentration of tRNA species capable of decoding them, which do not always correlate. Here, we developed a metric to predict organism-specific relative translation rates of codons based on the availability of tRNA decoding mechanisms: Watson-Crick, non-Watson-Crick or both types of interactions. We determine translation rates of messages by pulse-chase analyses in living Escherichia coli cells and show that sequence engineering based on these concepts predictably modulates translation rates in a manner that is superior to codon usage frequency, which occur during the elongation phase, and significantly impacts folding of the encoded polypeptide. Finally, we demonstrate that sequence harmonization based on expression host tRNA pools, designed to mimic ribosome movement of the original organism, can significantly increase the folding of the encoded polypeptide. These results illuminate how genetic code degeneracy may function to specify properties beyond amino acid encoding, including folding.

The neurodegenerative-disease-related protein sacsin is a molecular chaperone.

Various human neurodegenerative disorders are associated with processes that involve misfolding of polypeptide chains. These so-called protein misfolding disorders include Alzheimer's and Parkinson's diseases and an increasing number of inherited syndromes that affect neurons involved in motor control circuits throughout the central nervous system. The reasons behind the particular susceptibility of neurons to misfolded proteins are currently not known. The main function of a class of proteins known as molecular chaperones is to prevent protein misfolding and aggregation. Although neuronal cells contain the major known classes of molecular chaperones, central-nervous-system-specific chaperones that maintain the neuronal proteome free from misfolded proteins are not well defined. In this study, we assign a novel molecular chaperone activity to the protein sacsin responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay, a degenerative disorder of the cerebellum and spinal cord. Using purified components, we demonstrate that a region of sacsin that contains a segment with homology to the molecular chaperone Hsp90 is able to enhance the refolding efficiency of the model client protein firefly luciferase. We show that this region of sacsin is highly capable of maintaining client polypeptides in soluble folding-competent states. Furthermore, we demonstrate that sacsin can efficiently cooperate with members of the Hsp70 chaperone family to increase the yields of correctly folded client proteins. Thus, we have identified a novel chaperone directly involved in a human neurodegenerative disorder.

The sacsin repeating region (SRR): a novel Hsp90-related supra-domain associated with neurodegeneration.

Protein supra-domains are defined as recurring arrangements of two or three domains present adjacent to each other along a polypeptide chain. Such combinations have novel functions beyond those of the individual partner domains that compose them, which can exist in isolation. Here, we describe a new type of large supra-domain (approximately 360 residues) in which one of the component partners (approximately 200 residues) appears to be incapable of existing in a context other than immediately adjacent to the C-terminus of the well-characterized Hsp90-like ATPase domain. We found that this supra-domain has a broad phylogenetic distribution, with examples in Archaea, Bacteria, and Eukarya. There is strong selective pressure for this arrangement to occur as part of repeated regions of unprecedented length. We identified multiple strategies of convergent evolution to attain such configurations. In humans, this supra-domain is present in triplicate at the N-terminus of the protein sacsin (4579 residues), mutated in the neurodegenerative disorder known as spastic ataxia of Charlevoix-Saguenay, and thus, we termed it "sacsin repeating region" (SRR). Biochemical characterization demonstrated that SRRs possess ATPase activity, which appears to be a requirement for sacsin function, as a disease-causing mutation leads to an alternate conformation completely incapable of hydrolyzing ATP. We also found evidence of a convergent evolutionary strategy to place SRRs in proteins containing C-terminal J domains, which we demonstrated here to be capable of stimulating the intrinsic ATPase activity of Hsp70. Our sequence and biochemical analyses indicate that SRRs necessitate nucleotide hydrolysis for their function, provided by the common Hsp90 ATPase domain, which, when coupled to the unique adjacent sequence, may give rise to a novel activity related to protein quality control.

Slowing bacterial translation speed enhances eukaryotic protein folding efficiency.

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.

Urinary side effects and complications after permanent prostate brachytherapy: the MD Anderson Cancer Center experience.

OBJECTIVES: To evaluate acute and long-term urinary morbidity after permanent prostate brachytherapy at a single tertiary care center. To minimize the risk of long-term urinary morbidity, it is important for clinicians to be able to distinguish acute urinary side effects after prostate brachytherapy from longer-term treatment-related urinary complications. METHODS: The medical records of 351 consecutive patients who underwent prostate brachytherapy at the MD Anderson Cancer Center between 1998 and 2006 were analyzed. To evaluate the short-term urinary side effects, the Expanded Prostate Cancer Index Composite questionnaire was administered at baseline and at 1, 4, 8, and 12 months. Long-term urinary complications were scored using a modified Radiation Therapy Oncology Group scale. RESULTS: All 4 urinary subdomain scores evaluating acute urinary side effects after treatment (bother, function, incontinence, and irritation or obstruction) had returned to baseline levels by 8 months after implantation. At 5 years, the cumulative risks of late urinary complications by grade were 8.6% for grade 1 complications, 6.5% for grade 2, 1.7% for grade 3%, and 0.5% for grade 4. The most common grade 2 late urinary complications were urethral stricture (4 patients), incontinence requiring daily pads (3 patients), and intermittent hematuria (3 patients). Grade 3 complications were urinary retention requiring self-catheterization (2 patients) and severe frequency with dysuria (2 patients). The only grade 4 event was severe hemorrhagic cystitis. CONCLUSIONS: Short-term urinary side effects after prostate brachytherapy are common, follow a predictable course, and typically resolve within 1 year. Conservative management of short-term urinary side effects is recommended to minimize the risk of long-term urinary complications.

Toll-like receptor 7 mitigates lethal West Nile encephalitis via interleukin 23-dependent immune cell infiltration and homing.

West Nile virus (WNV), a mosquito-transmitted single-stranded RNA (ssRNA) flavivirus, causes human disease of variable severity. We investigated Toll-like receptor 7-deficient (Tlr7(-/-)) and myeloid differentiation factor 88-deficient (Myd88(-/-)) mice, which both have defective recognition of ssRNA, and found increased viremia and susceptibility to lethal WNV infection. Despite increased tissue concentrations of most innate cytokines, CD45(+) leukocytes and CD11b(+) macrophages failed to home to WNV-infected cells and infiltrate into target organs of Tlr7(-/-) mice. Tlr7(-/-) mice and macrophages had reduced interleukin-12 (IL-12) and IL-23 responses after WNV infection, and mice deficient in IL-12 p40 and IL-23 p40 (Il12b(-/-)) or IL-23 p19 (Il23a(-/-)), but not IL-12 p35 (Il12a(-/-)), responded similarly to Tlr7(-/-) mice, with increased susceptibility to lethal WNV encephalitis. Collectively, these results demonstrate that TLR7 and IL-23-dependent WNV responses represent a vital host defense mechanism that operates by affecting immune cell homing to infected target cells.

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells.

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.

Role of two distinct gammadelta T cell subsets during West Nile virus infection.

gammadelta T cells respond rapidly following West Nile virus (WNV) infection, limiting viremia and invasion of the central nervous system and thereby protecting the host from lethal encephalitis. Here, we investigated the role of two major subpopulations of peripheral gammadelta T cells, Vgamma1(+) and Vgamma4(+) cells, in host immunity against WNV infection. We found initially that aged mice were more susceptible to WNV infection than young mice. Following WNV challenge, Vgamma1(+) cells in young mice expanded significantly whereas Vgamma4(+) cells expanded modestly. In contrast, aged mice exhibited a slower and reduced response of Vgamma1(+) cells but maintained a higher content of Vgamma4(+) cells. Vgamma1(+) cells were the major gammadelta subset producing IFN-gamma during WNV infection. Mice depleted of Vgamma1(+) cells had an enhanced viremia and higher mortality to WNV encephalitis. Vgamma4(+) cells had a higher potential for producing tumor necrosis factor-alpha (TNF-alpha), a cytokine known to be involved in blood-brain barrier compromise and WNV entry into the brain. Depletion of Vgamma4(+) cells reduced TNF-alpha level in the periphery, accompanied by a decreased viral load in the brain and a lower mortality to WN encephalitis. These results suggest that Vgamma1(+) and Vgamma4(+) cells play distinct roles in protection and pathogenesis during WNV infection.

Four-dimensional computed tomography scan analysis of tumor and organ motion at varying levels of abdominal compression during stereotactic treatment of lung and liver.

PURPOSE: To investigate the effectiveness of different abdominal compression levels on tumor and organ motion during stereotactic body radiotherapy of lower lobe lung and liver tumors using four-dimensional (4D)-CT scan analysis. METHODS AND MATERIALS: Three 4D-CT scans were acquired for 10 patients first using with no compression and then compared with two different levels of abdominal compression. The position of the tumor and various organs were defined at the peak inspiratory and expiratory phases and compared to determine the maximum motion. RESULTS: Mean (+/-SD) medium compression force (MC) and high compression force (HC) were 47.6 +/- 16.0 N and 90.7 +/- 27.1 N, respectively. Mean overall tumor motion was 13.6 mm (2sigma [2 sigma] 11.5-15.6), 8.3 mm (2sigma 6.0-10.5), and 7.2 mm (2sigma 5.4-9.0) for no compression, MC, and HC, respectively. A significant difference in the control of both superior-inferior (SI) and overall motion of tumors was seen with the application of MC and HC when compared with no compression (p < 0.0001 for both). High compression force improved SI and overall tumor motion compared with MC, but this was only significant for SI motion (p = 0.04 and p = 0.06). Significant control of organ motion was only seen in the pancreas (p = 0.01). CONCLUSIONS: Four-dimensional CT shows significant control of both lower lobe lung and liver tumors using abdominal compression. High levels of compression improve SI tumor motion when compared with MC.

West nile virus attenuates activation of primary human macrophages.

West Nile virus (WNV) is a mosquito-borne flavivirus that has spread rapidly throughout the U.S. and there is currently no effective treatment. Understanding the pathogenesis of WNV infection in humans is critical for development of a potent therapy. In this study, we examined the activation of primary human macrophages in response to WNV infection, and showed that WNV interacts with human macrophages at multiple levels. While infection with WNV induced production of interleukin (IL)-8, production of IL-1beta, and type I interferon was inhibited. Infection with WNV interferes with the downstream JAK/STAT pathway, which is important for macrophage activation. In comparison to other related flaviviruses, the differential response of proinflammatory cytokines is distinct to WNV.

A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.

The tick Ixodes scapularis is an efficient vector for microbes, including the Lyme disease agent Borrelia burgdorferi. Ticks engorging on vertebrates induce recruitment of inflammatory cells to the bite site. For efficient transmission to the vector, pathogens have to traffic through this complex feeding site while avoiding the deleterious effects of immune cells. We show that a tick protein, Salp25D, plays a critical role-in the mammalian host-for acquisition of Borrelia burgdorferi by the vector. Silencing salp25D in tick salivary glands impaired spirochete acquisition by ticks engorging on B. burgdorferi-infected mice. Immunizing mice against Salp25D also decreased Borrelia acquisition by I. scapularis. Salp25D detoxified reactive oxygen species at the vector-pathogen-host interface, thereby providing a survival advantage to B. burgdorferi at the tick feeding site in mice. These data demonstrate that pathogens can exploit arthropod molecules to defuse mammalian responses in order to successfully enter the vector.

Antiviral peptides targeting the west nile virus envelope protein.

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

Isolations of Potosi virus from mosquitoes (Diptera: Culicidae) collected in Connecticut.

Potosi virus (POTV) (Bunyaviridae: Orthobunyavirus) was first isolated from Aedes albopictus (Skuse) collected in Potosi, MO, in 1989, and subsequent isolations were reported from Illinois, Michigan, Ohio, and the Carolinas. To determine whether the distribution of this virus extends into the northeastern United States, we analyzed arboviruses acquired from mosquitoes collected in Connecticut from 1998 to 2004. In 2001, a bunyavirus was isolated from Aedes vexans (Meigen) that was different from other arboviruses known to occur in Connecticut by cross-neutralization and reverse transcription-polymerase chain reaction (RT-PCR) assays. Nucleotide and encoded amino acid sequences of a portion of the G2 envelope gene were 99 and 100% similar to POTV, respectively, yet distinct from indigenous strains of Jamestown Canyon (JCV), Cache Valley (CVV), and Trivittatus virus (TVTV). Viral isolates obtained from the statewide surveillance program were retested by RT-PCR coupled with restriction enzyme analysis to distinguish POTV from other bunyaviruses. POTV isolates, previously typed by neutralization, were correctly identified by RT-PCR; however, many isolates classified as JCV or CVV by enzyme-linked immunosorbent assay proved to be POTV by molecular assays. In total, 92 strains of POTV were isolated from 12 mosquito species in 2000, 2001, and 2003, whereas POTV was not detected in mosquitoes sampled during 1998, 1999, 2002, and 2004. Viral isolation rates were highest for Anopheles punctipennis (Say) (3.2-11.3 infection rate per 1,000 mosquitoes), whereas the greatest number of isolates came from Ochlerotatus trivittatus (Coquillett) (8-16 isolates). This finding represents the first detection of POTV in the northeastern United States where it infects a diverse array of mosquito species.