Point Absorber Limits to Future Gravitational-Wave Detectors.

High-quality optical resonant cavities require low optical loss, typically on the scale of parts per million. However, unintended micron-scale contaminants on the resonator mirrors that absorb the light circulating in the cavity can deform the surface thermoelastically and thus increase losses by scattering light out of the resonant mode. The point absorber effect is a limiting factor in some high-power cavity experiments, for example, the Advanced LIGO gravitational-wave detector. In this Letter, we present a general approach to the point absorber effect from first principles and simulate its contribution to the increased scattering. The achievable circulating power in current and future gravitational-wave detectors is calculated statistically given different point absorber configurations. Our formulation is further confirmed experimentally in comparison with the scattered power in the arm cavity of Advanced LIGO measured by in situ photodiodes. The understanding presented here provides an important tool in the global effort to design future gravitational-wave detectors that support high optical power and thus reduce quantum noise.

Approaching the motional ground state of a 10-kg object.

The motion of a mechanical object, even a human-sized object, should be governed by the rules of quantum mechanics. Coaxing them into a quantum state is, however, difficult because the thermal environment masks any quantum signature of the object's motion. The thermal environment also masks the effects of proposed modifications of quantum mechanics at large mass scales. We prepared the center-of-mass motion of a 10-kilogram mechanical oscillator in a state with an average phonon occupation of 10.8. The reduction in temperature, from room temperature to 77 nanokelvin, is commensurate with an 11 orders-of-magnitude suppression of quantum back-action by feedback and a 13 orders-of-magnitude increase in the mass of an object prepared close to its motional ground state. Our approach will enable the possibility of probing gravity on massive quantum systems.

Quantum-Enhanced Advanced LIGO Detectors in the Era of Gravitational-Wave Astronomy.

The Laser Interferometer Gravitational Wave Observatory (LIGO) has been directly detecting gravitational waves from compact binary mergers since 2015. We report on the first use of squeezed vacuum states in the direct measurement of gravitational waves with the Advanced LIGO H1 and L1 detectors. This achievement is the culmination of decades of research to implement squeezed states in gravitational-wave detectors. During the ongoing O3 observation run, squeezed states are improving the sensitivity of the LIGO interferometers to signals above 50 Hz by up to 3 dB, thereby increasing the expected detection rate by 40% (H1) and 50% (L1).

Effects of transients in LIGO suspensions on searches for gravitational waves.

This paper presents an analysis of the transient behavior of the Advanced LIGO (Laser Interferometer Gravitational-wave Observatory) suspensions used to seismically isolate the optics. We have characterized the transients in the longitudinal motion of the quadruple suspensions during Advanced LIGO's first observing run. Propagation of transients between stages is consistent with modeled transfer functions, such that transient motion originating at the top of the suspension chain is significantly reduced in amplitude at the test mass. We find that there are transients seen by the longitudinal motion monitors of quadruple suspensions, but they are not significantly correlated with transient motion above the noise floor in the gravitational wave strain data, and therefore do not present a dominant source of background noise in the searches for transient gravitational wave signals. Using the suspension transfer functions, we compared the transients in a week of gravitational wave strain data with transients from a quadruple suspension. Of the strain transients between 10 and 60 Hz, 84% are loud enough that they would have appeared above the sensor noise in the top stage quadruple suspension monitors if they had originated at that stage at the same frequencies. We find no significant temporal correlation with the suspension transients in that stage, so we can rule out suspension motion originating at the top stage as the cause of those transients. However, only 3.2% of the gravitational wave strain transients are loud enough that they would have been seen by the second stage suspension sensors, and none of them are above the sensor noise levels of the penultimate stage. Therefore, we cannot eliminate the possibility of transient noise in the detectors originating in the intermediate stages of the suspension below the sensing noise.

Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity.

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.

Entomological studies along the Colorado Front Range during a period of intense West Nile virus activity.

To better understand the ecology of West Nile virus transmission in Northern Colorado, field studies were conducted in Larimer and Weld counties from September 2003 through March 2005. During summer studies, 18,540 adult mosquitoes were collected using light traps and gravid traps. West Nile virus RNA was detected in 24 of the 2,140 mosquito pools tested throughout the study area in 2003 and 2004. Culex tarsalis had the highest minimum infection rate (MIR) in both 2003 (MIR = 34.48) and in 2004 (MIR = 8.74). During winter studies, 9,391 adult mosquitoes were collected by aspirator from various overwintering sites including bridges and storm drains. The most frequently collected species was Culex pipiens. West Nile virus was not detected in our overwintering collections. The relationship between spring adult emergence and temperature inside and outside overwintering sites is described. Species composition of collections as well as the spatial and temporal distribution of West Nile virus detections are presented.

Effect of La Crosse virus infection on insemination rates in female Aedes triseriatus (Diptera: Culicidae).

Aedes triseriatus (Say) (Diptera: Culicidae) females orally infected with La Crosse virus after ingesting an infectious bloodmeal were compared for mating efficiency with females that ingested a noninfectious bloodmeal. After 14-d extrinsic incubation to allow for dissemination of the infection, all females were offered a second noninfectious bloodmeal and were placed in cages with age-matched males for 5 d. After 6 d, insemination rates were determined by detection of sperm in the spermathecae. Insemination rates of the La Crosse virus-infected females were significantly greater than in uninfected females.

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes.

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.

Sindbis virus-induced silencing of dengue viruses in mosquitoes.

Aedes aegypti were injected intrathoracically with double subgenomic Sindbis (dsSIN) viruses with inserted sequences derived from the genome of one or more of the four dengue (DEN) virus serotypes. Mosquitoes were highly resistant to challenge with homologous DEN viruses from which the effector sequences were derived, and resistance to DEN viruses was independent of the orientation of the effector RNA. dsSIN viruses designed to express RNA derived from the premembrane coding region of DEN-2 prevented the accumulation of DEN2 RNA, and C6/36 cells were highly resistant to DEN-2 virus when challenged at 2, 5 or 8 days after the initial dsSIN virus infections, even though the dsSIN-derived RNA had sharply declined at the later time points. Initiation of resistance occurred prior to or within the first 8 h after challenge with DEN-2 virus. We conclude that DEN viruses are inhibited by a mechanism similar to post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) phenomena described in plants and invertebrates, respectively. The potential occurrence of PTGS or RNAi in mosquitoes and mosquito cells suggests new ways of inhibiting the replication of arthropod-borne viruses in mosquito vectors, studying vector-virus interactions, and silencing endogenous mosquito genes.

Identification and sequence determination of mRNAs detected in dormant (diapausing) Aedes triseriatus mosquito embryos.

Many insects survive adverse climatic conditions in a dormant state known as diapause. In this study, we identified and sequenced several mRNAs in diapausing Aedes triseriatus mosquito embryos. Using reverse-transcription PCR and 5' RACE, we identified a 995-nucleotide cDNA that encodes a 259-amino acid protein of unknown function. This putative protein displays strong sequence similarity to Drosophila melanogaster (95%), human (87%), Caenorhabditis elegans (86%) and yeast (81%) counterparts. The second identified full-length cDNA consists of 624 nucleotides and encodes a 174-amino acid protein of unknown function. This putative protein displays significant sequence similarity to D. melanogaster (68%), human (59%), plant (57%) and yeast (49%) counterparts. We also detected a number of cDNA fragments that exhibited significant sequence similarity to a mitochondrial cytochrome C oxidase subunit, human N33 protein (a potential human prostate tumor suppressor), 18S and 28S ribosomal RNAs, protein disulfide-isomerase, and guanine nucleotide-binding protein.

Complete cDNA and deduced amino acid sequence of the chaperonin containing T-complex polypeptide 1 (CCT) delta subunit from Aedes triseriatus mosquitoes.

The chaperonin containing t-complex polypeptide 1 (CCT) assists in the ATP-dependent folding and assembly of newly translated actin and tubulin in the eukaryotic cytosol. CCT is composed of eight different subunits, each encoded by an independent gene. In this report, we used RT-PCR amplification and 5'- and 3'-rapid amplification of cDNA ends (RACE) to determine the complete cDNA sequence of the CCT delta subunit from Aedes triseriatus mosquitoes. The CCT delta cDNA is 1936 nucleotides in length and encodes a putative 533 amino acid protein with a calculated molecular mass of 57,179 daltons and pI of 7.15. Hydrophobic residues comprise 39.8% of the amino acid sequence and putative motifs for ATP-binding and ATPase-activity are present. The amino acid sequence displays strong sequence similarity to Drosophila melanogaster (92%), human (85%), puffer fish (84%) and mouse (84%) counterparts. CCT delta mRNA was detected in both biosynthetically active (embryonating) and dormant (diapausing) Ae. triseriatus embryos by RT-PCR analysis.

Antibody prophylaxis and therapy for flavivirus encephalitis infections.

The outbreak of West Nile (WN) encephalitis in the United States has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. Although equine WN vaccines are currently being developed, a WN vaccine for humans is years away. There is also no specific therapeutic agent for flaviviral infections. The incidence of human WN virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the vaccine's economic feasibility. It has been shown, however, that prophylactic application of antiflaviviral antibody can protect mice from subsequent virus challenge. This model of antibody prophylaxis using murine monoclonal antibodies (MAbs) has been used to determine the timing of antibody application and specificity of applied antibody necessary for successful prophylaxis. The major flaviviral antigen is the envelope (E) glycoprotein that binds cellular receptors, mediates cell membrane fusion, and contains an array of epitopes that elicit virus-neutralizing and nonneutralizing antibodies. The protective efficacy of an E-glycoprotein-specific MAb is directly related to its ability to neutralize virus infectivity. The window for successful application of prophylactic antibody to prevent flaviviral encephalitis closes at about 4 to 6 days postinfection concomitant with viral invasion of the brain. Using murine MAbs to modify human disease results in a human antimouse antibody (HAMA) response that eventually limits the effectiveness of subsequent murine antibody applications. To reduce the HAMA response and make these MAbs more generally useful for humans, murine MAbs can be "humanized" or human MAbs with analogous reactivities can be developed. Antiflaviviral human or humanized MAbs might be practical and cost-effective reagents for preventing or modifying flaviviral diseases.

Molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes.

Arthropod-borne virus (arbovirus) infections cause a number of emerging and resurgent human and veterinary infectious diseases. Traditional means of controlling arbovirus diseases include vaccination of susceptible vertebrates and mosquito control, but in many cases these have been unavailable or ineffective, and so novel strategies for disease control are needed. One possibility is genetic manipulation of mosquito vectors to render them unable to transmit arboviruses. This review describes recent work to test the concept of pathogen-derived resistance in arthropods by expression of viral genes in mosquito cell cultures and mosquitoes. Sense and antisense genome sequences from La Crosse virus (LAC) (a member of the Bunyaviridae) and dengue viruses serotypes 1 to 4 (DEN-1 to DEN-4) (members of the Flaviviridae) were expressed in mosquito cells from double-subgenomic and replicon vectors based on Sindbis virus (a member of the Togaviridae). The cells were then challenged with homologous or related viruses. For LAC, expression of antisense sequences from the small (S) genome segment, particularly full-length antisense S RNA, effectively interfered with replication of challenge virus, whereas expression of either antisense or sense RNA from the medium (M) segment was completely ineffective in LAC inhibition. Expression of sense and antisense RNA derived from certain regions of the DEN genome also blocked homologous virus replication more effectively than did RNA from other regions. Other parameters of RNA-mediated interference have been defined, such as the time when replication is blocked and the minimum size of effector RNA. The mechanism of RNA inhibition has not been determined, although it resembles double-stranded RNA interference in other nonvertebrate systems. Prospects for application of molecular strategies to control arbovirus diseases are briefly reviewed.

Molecular cloning and complete cDNA sequences of the ribosomal proteins rpl34 and rpl44 from Aedes triseriatus mosquitoes.

We present the complete cDNA and deduced amino acid sequences of the 60S ribosomal subunit proteins, rpL34 and rpL44, from Aedes triseriatus mosquitoes. The rpL34 cDNA is 554 nucleotides in length and encodes a 139 amino acid protein with a calculated molecular mass of 15 732 daltons. The putative protein displays strong sequence similarity to rpL34 of Aedes albopictus mosquitoes (92%), humans (60%) and rats (58%). The protein is highly basic and contains a C-terminal repetitive-alanine domain and four putative nucleolar localization signals. The rpL44 cDNA consists of 450 nucleotides and encodes a 104 amino acid protein with a calculated molecular mass of 12 544 daltons. The putative protein displays strong sequence similarity to rpL44 of Brugia malayi (87%), Caenorhabditis elegans (86%) and humans (85%). The protein is highly basic and contains a putative nucleolar localization signal. The mRNAs for both rpL34 and rpL44 were detected in biosynthetically active (embryonating) and dormant (diapausing) Ae. triseriatus embryos by RT-PCR analysis.

La Crosse virus infection of Aedes triseriatus (Diptera: Culicidae) ovaries before dissemination of virus from the midgut.

La Crosse (LAC) virus is efficiently transmitted transovarially by the mosquito Aedes triseriatus (Say). To determine the time course and tropisms of LAC virus infection of ovaries, immunofluorescent antibody staining, in situ hybridization, and reverse transcription-polymerase chain reaction techniques were used to detect viral antigen and RNA in the ovaries. LAC virus was detected in the ovaries (presumably in calyx tissues) by all 3 assays at day 2 after infection and before dissemination from the midgut on day 6. Apparently, ovaries can become infected by mechanisms other than by dissemination of virus from a midgut infection. By days 8-14 after infection, virus analytes became detectable in many tissues within the ovary including follicular epithelium, oocytes, nurse cells, and calyx, reflecting the remarkable host parasite relationship between LAC virus and its mosquito vector.

Short report: Diapause, transovarial transmission, and filial infection rates in geographic strains of La Crosse virus-infected Aedes triseriatus.

La Crosse (LAC) virus is transmitted horizontally to vertebrates and vertically to progeny by Aedes triseriatus mosquitoes, and in northern midwestern states, this virus overwinters in diapausing eggs of the vector. In Florida, the vector remains active throughout the year and does not diapause. To determine if there is an association between diapause and vertical transmission efficiency of LAC virus, transovarial transmission (TOT), and filial infection (FI) rates were determined for geographic strains of Ae. triseriatus. The TOT rates were not significantly different for Ae. triseriatus originating from Florida (78%) and those from Wisconsin (85%). The FI rates did differ significantly between the two groups (33% and 45%, respectively, for the Florida and Wisconsin mosquitoes). Furthermore, a line of mosquitoes was selected from a Wisconsin colony that had a reduced diapause phenotype (the AD- strain). While this strain displayed TOT rates that were the same as the other Wisconsin mosquitoes (85%), the FI rates were significantly lower (34%), indicating a reduction in TOT efficiency. The role of vertical transmission capacity in LAC virus endemicity remains to be determined.

Engineered resistance in Aedes aegypti to a West African and a South American strain of yellow fever virus.

Double subgenomic Sindbis (dsSIN) viruses were engineered to transduce mosquito cells with antisense RNA derived either from the premembrane (prM) or polymerase (NS5) coding regions of the 17D vaccine strain of yellow fever virus (YFV). Aedes albopictus C6/36 cells were infected at high multiplicities of infection (MOI) with each dsSIN virus. Forty-eight hours later, the transduced cells were challenged with an MOI of 0.1 of the Asibi strain of YFV. At 72-hr postchallenge, the cells were assayed by immunofluorescence for the presence of YFV antigen. Cells transduced with prM or NS5 antisense RNAs derived from the YFV genome displayed no YFV-specific antigens. In contrast, cells infected with control dsSIN viruses that expressed no antisense RNA or dengue virus-derived antisense RNAs were permissive for the challenge virus. To analyze resistance in the mosquito, five log10 50% tissue culture infective doses (TCID50) of each dsSIN virus and three log10TCID50 of either a West African (BA-55) or South American (1899/81) strain of wild-type YFV were coinoculated into Ae. aegypti. Mosquitoes transduced with effector RNAs targeting the prM or NS5 gene regions did not transmit West African YFV and poorly transmitted the South American strain of YFV.

Effect of La Crosse virus infection on overwintering of Aedes triseriatus.

The effect of La Crosse (LAC) virus infection on Aedes triseriatus overwintering success was determined. Eggs from LAC virus transovarially infected (LAC TOT+) and uninfected (LAC TOT-) Ae. triseriatus colonies were induced into diapause, held in natural conditions, and returned to the laboratory at predetermined times for assay of diapause, mortality, and filial infection rates, and to examine viral transcription and replication during diapause. Embryos from the LAC TOT+ colony exhibited greater cumulative mortality (16.7%) than the LAC TOT- eggs (7.3%) throughout the overwintering periods. The increased mortality rate in LAC TOT+ eggs corresponded with a decrease in filial infection rates. Eggs from the LAC TOT+ colony terminated diapause more readily than the LAC TOT- colony. An RNA strand-specific reverse transcriptase-polymerase chain reaction technique was used to monitor viral transcription and replication in mosquito eggs during overwintering, and to compare viral replication in diapausing and nondiapausing embryos. Viral messenger and replicative form RNA were present in eggs in all sample periods, suggesting that some virus replication occurred during diapause.

Analysis of LaCrosse virus S mRNA 5' termini in infected mosquito cells and Aedes triseriatus mosquitoes.

Nucleotide sequences were determined for the 5' termini of La Crosse virus (LAC) S segment mRNA from persistently infected mosquito cell cultures (C6/36 from Aedes albopictus) and embryos (Aedes triseriatus). LAC primes transcription of its mRNA with "scavenged" 5' caps and adjacent oligonucleotides from host mRNAs, and these non-virus-encoded 5'-terminal extensions are heterogeneous in infected mammalian cells. The nature of mosquito host-derived primers has not been previously investigated. During early C6/36 cell infection, LAC mRNA 5'-terminal sequences were heterogeneous, but variability decreased as infection persisted. One predominant sequence, 5' CCACTCGCCACT (sequence 1), was observed throughout C6/36 cell infection but was more prevalent after 15 days postinfection. This LAC mRNA 5'-terminal sequence comprised 81% of the scavenged host oligonucleotides from vertically infected A. triseriatus eggs during embryogenesis. As these embryos progressed in the dormant overwintering stage (diapause), the predominant scavenged sequence became 5' AGGAAAAGATGGT (sequence 2), and sequence 1 became less prevalent. As the eggs emerged from diapause, the LAC mRNA 5' termini were more variable; 33% had sequence 1, and the remainder were heterogeneous. In post-diapausing eggs, 100% of viral mRNAs had sequence 1 at their 5' termini. Molecular analyses thus revealed continuous but selective LAC cap scavenging during persistent C6/36 cell infection and during embryogenesis and diapause in A. triseriatus eggs. The variety of host-derived sequences was limited in both biosynthetically active (embryonating) and dormant (diapausing) eggs.

Analysis of La Crosse virus S-segment RNA and its positive-sense transcripts in persistently infected mosquito tissues.

La Crosse (LAC) virus is an important cause of pediatric arboviral encephalitis in the United States. LAC virus is biologically transmitted by the mosquito Aedes triseriatus, and, like other arthropod-borne viruses, it establishes a persistent, nonpathogenic infection in its vector following oral infection. To investigate LAC virus persistent infection of mosquitoes, a reverse transcription-PCR assay was developed for the amplification of LAC virus negative-sense small (S) genome RNA segment, its full-length complement, and its mRNA transcript for qualitative analysis of transcription and replication in persistently infected mosquito tissues. RNAs were assayed from midguts removed at predetermined times after infection with a LAC virus-containing blood meal. LAC virus genome was detected almost uniformly in midguts at days 3 to 28 postinfection (p.i.) and, as the time p.i. progressed, in more of the samples than either mRNA or viral cRNA (vcRNA). Thus, persistent LAC virus infection of A. triseriatus midguts was correlated with a reduction in detectable viral mRNA and vcRNA. The assay was also used for analysis of virus-specified RNA in both quiescent and biosynthetically active mosquito ovaries. Viral replication decreased, as indicated by the absence of viral mRNA and vcRNA, in the ovaries of mosquitoes that did not receive further blood meals after their original oral infection. Viral replication increased in ovaries of mosquitoes that took an additional blood meal 30 days p.i. and was continuous in mosquitoes that took multiple meals to stimulate oogenesis. Thus, virus replication in persistently infected mosquito ovaries was dependent on host cell biosynthetic status.