Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

The strain of West Nile virus (WNV) currently epidemic in North America contains a genetic mutation elevating its virulence in birds, especially species in the family Corvidae. Although dead American Crows (Corvus brachyrhynchos) have been the hallmark of the epidemic, the overall impact of WNV on North America's avifauna remains poorly understood and has not been addressed thoroughly in California. Here, we evaluate variation by species in the effect of WNV on California birds from 2004 to 2007 by using (1) seroprevalence in free-ranging birds, (2) percentage of carcasses of each species reported by the public that tested positive for WNV, (3) mortality determined from experimental infections, and (4) population declines detected by trend analysis of Breeding Bird Survey (BBS) data. Using Bayesian linear models, we extrapolate trends in BBS data from 1980 to . We attribute significant declines from expected abundance trends in areas supporting epiornitics to WNV transmission. We combine risk assessed from each of the four data sets to generate an overall score describing WNV risk by species. The susceptibility of California avifauna to WNV varies widely, with overall risk scores ranging from low for the refractory Rock Pigeon (Columba livia) through high for the susceptible American Crow. Other species at high risk include, in descending order, the House Finch (Carpodacus mexicanus), Black-crowned Night-Heron (Nycticorax nycticorax), Western Scrub-Jay (Aphelocoma californica), and Yellow-billed Magpie (Pica nuttalli). Our analyses emphasize the importance of multiple data sources in assessing the effect of an invading pathogen.

Host selection by vector mosquitoes is a critical component of virus proliferation, particularly for viruses such as West Nile (WNV) that are transmitted enzootically to a variety of avian hosts, and tangentially to dead-end hosts such as humans. Culex tarsalis is a principal vector of WNV in rural areas of western North America. Based on previous work, Cx. tarsalis utilizes a variety of avian and mammalian hosts and tends to feed more frequently on mammals in the late summer than during the rest of the year. To further explore this and other temporal changes in host selection, bloodfed females were collected at a rural farmstead and heron nesting site in Northern California from May 2008 through May 2009, and bloodmeal hosts identified using either a microsphere-based array or by sequencing of the mitochondrial cytochrome c oxidase I (COI) gene. Host composition during summer was dominated by four species of nesting Ardeidae. In addition, the site was populated with various passerine species as well as domestic farm animals and humans. When present, Cx. tarsalis fed predominantly (>80%) upon the ardeids, with Black-crowned Night-Herons, a highly competent WNV host, the most prevalent summer host. As the ardeids fledged and left the area and mosquito abundance increased in late summer, Cx. tarsalis feeding shifted to include more mammals, primarily cattle, and a high diversity of avian species. In the winter, Yellow-billed Magpies and House Sparrows were the predominant hosts, and Yellow-billed Magpies and American Robins were fed upon more frequently than expected given their relative abundance. These data demonstrated that host selection was likely based both on host availability and differences in utilization, that the shift of bloodfeeding to include more mammalian hosts was likely the result of both host availability and increased mosquito abundance, and that WNV-competent hosts were fed upon by Cx. tarsalis throughout the year.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective "avian herd immunity" due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003-2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble's quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003-2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV.

The invasion of different southern California landscapes by West Nile virus (WNV) and its subsequent amplification to epidemic levels during 2004 enabled us to study the impact of differing corvid populations in three biomes: the hot Colorado desert with few corvids (Coachella Valley), the southern San Joaquin Valley (Kern County) with large western scrub-jay but small American crow populations, and the cool maritime coast (Los Angeles) with a large clustered American crow population. Similar surveillance programs in all three areas monitored infection rates in mosquitoes, seroconversion rates in sentinel chickens, seroprevalence in wild birds, numbers of dead birds reported by the public, and the occurrence of human cases. Infection rates in Culex tarsalis Coquillett and sentinel chicken seroconversion rates were statistically similar among all three areas, indicating that highly competent mosquito hosts were capable of maintaining enzootic WNV transmission among less competent and widely distributed avian hosts, most likely house sparrows and house finches. In contrast, infection rates in Culex pipiens quinquefasciatus Say were statistically higher in Kern and Los Angeles counties with elevated corvid populations than in Coachella Valley with few corvids. Spatial analyses of dead corvids showed significant clusters near known American crow roosts in Los Angeles that were congruent with clusters of human cases. In this area, the incidence of human and Cx. p. quinquefasciatus infection was significantly greater within corvid clusters than without, indicating their importance in virus amplification and as a risk factor for human infection. In contrast the uniform dispersion by territorial western scrub-jays resulted in a high, but evenly distributed, incidence of human disease in Kern County.

Abstract. To determine whether West Nile virus (WNV) persistent infection in avian hosts may potentially serve as an overwintering mechanism, House Sparrows and House Finches, experimentally and naturally infected with several strains of WNV, and two naturally infected Western Scrub-Jays were held in mosquito-proof outdoor aviaries from 2007-March 2008. Overall, 94% (n = 36) of House Sparrows, 100% (n = 14) of House Finches and 2 Western Scrub-Jays remained WNV antibody positive. When combined by species, 37% of the House Sparrows, 50% of the House Finches, and 2 Western Scrub-Jays were WNV RNA positive at necropsy, up to 36 weeks post-infection. Infectious WNV was not detected. Our study supports the hypothesis that some avian hosts support the long-term persistence of WNV RNA, but it remains unresolved whether these infections relapse to restart an avian-arthropod transmission cycle and thereby serve as an overwintering mechanism for WNV.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective "avian herd immunity" due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003-2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble's quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003-2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV.

Campylobacter jejuni is a foodborne pathogen that often leads to human infections through the consumption of contaminated poultry. Wild birds may play a role in the transmission of C. jejuni by acting as reservoir hosts. Despite ample evidence that wild birds harbor C. jejuni, few studies have addressed the role of host ecology in transmission to domestic animals or humans. We tested the hypothesis that host social behavior and habitat play a major role in driving transmission risk. C. jejuni infection and host ecology were studied simultaneously in wild American crows (Corvus brachyrhynchos) in Davis, CA, over 3 years. We found that 178 of 337 samples tested were culture positive (53%), with infection varying by season and host age. Among adult crows, infection rates were highest during the winter, when migrants return and crows form large communal roosts. Nestlings had the highest risk of infection, and whole-genome sequencing supports the observation of direct transmission between nestlings. We deployed global positioning system (GPS) receivers to quantify habitat use by crows; space use was nonrandom, with crows preferentially occupying some habitats while avoiding others. This behavior drastically amplified the risk of environmental contamination from feces in specific locations. This study demonstrates that social behavior contributes to infection within species and that habitat use leads to a heterogeneous risk of cross-species transmission. IMPORTANCECampylobacter jejuni is the most common cause of gastroenteritis in industrialized countries. Despite efforts to reduce the colonization of poultry flocks and eventual infection of humans, the incidence of human C. jejuni infection has remained high. Because wild birds can harbor strains of C. jejuni that eventually infect humans, there has long been speculation that wild birds might act as an important reservoir in the C. jejuni infection cycle. We simultaneously studied infection prevalence, social behavior, and movement ecology in wild American crows (Corvus brachyrhynchos). We found that social behavior contributed to patterns of infection and that movement behavior resulted in some areas having a high risk of transmission while others had a low risk. The incorporation of ecological data into studies of C. jejuni in wild birds has the potential to resolve when and how wild birds contribute to domestic animal and human C. jejuni infection, leading to better control of initial poultry contamination. Campylobacter jejuni is the leading cause of gastroenteritis in industrialized countries (1, 2), with most infections in humans resulting from the consumption of contaminated and improperly cooked poultry (3). Generally, infected individuals recover in a few days, but in some cases, infection can lead to hospitalization (ϳ15% of culture-positive cases in the United States [1]), chronic autoimmune disorders (ϳ2% of cases [4,5]), and even death (ϳ0.06% of cases [1]). These health concerns demand that food producers manage flock infection and disinfect sus...