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Many countries operate public health surveillance systems to monitor foodborne disease occurrence. The illness counts for pathogens of interest are monitored across time to assess if changes have occurred in case rates or the overall illness burden. Common to all of these systems is that only a fraction of all foodborne illnesses are reported and the relationship between observed and the total number of illnesses is uncertain. Food-safety policies intend to affect both the total and observed number of illnesses. Ideally, the surveillance system would be sufficiently sensitive to detect these changes, but the statistical power of these systems is generally not well understood. This study proposes two approaches for estimating the power of a foodborne illness surveillance system. These methods are then applied to assess the power of detecting specified changes in the total number of illnesses in an existing surveillance system. The findings suggest that the power of a national foodborne disease surveillance system to detect modest annual reductions in Salmonella illnesses may be limited. For example, a naive presumptive model that assumes observed illnesses are reduced directly by 10% predicts that the power to discern this difference, from one year to the next, is 0.21. A Bayesian model, that accounts for uncertainty in projecting changes in total illnesses to the number of observed illnesses, predicts that the power to detect a true 10% reduction is 0.04 (i.e., 4% confidence of detecting this magnitude of reduction or 96% chance of a Type II error) one year after the change has taken effect. Although the power of a national surveillance system increases as the magnitude of reductions increases – or as the number of years the reduction is maintained increases – the Bayesian model demonstrates that power is less than 0.5 for reductions up to 50% for one year and is less than 0.53 for reductions of 30% that are maintained for four years. The limited power to detect intended changes in total annual illnesses of national public health surveillance systems may highlight the need for regulators to also monitor food contamination evidence to gauge progress towards achieving intended policy effects.

Human activities are altering the fundamental geography of biogeochemicals. Yet we lack an understanding of how the spatial patterns in organismal stoichiometry affect biogeochemical processes and the tools to predict the impacts of global changes on biogeochemical processes. In this contribution we develop stoichiometric distribution models (StDMs), which allow us to map spatial structure in resource elemental composition across a landscape and evaluate spatial responses of consumers. We parameterise StDMs for a consumer-resource (moose-white birch) system and demonstrate that we can develop predictive models of resource stoichiometry across a landscape and that such models could improve our predictions of consumer space use. With results from our study system application, we argue that explicit consideration of the spatial patterns in organismal elemental composition may uncover emergent individual, population, community and ecosystem properties that are not revealed at the local extents routinely used in ecological stoichiometry. We discuss perspectives for further developments and application of StDMs to advance three emerging frameworks for spatial ecosystem ecology in an era of global change; meta-ecosystem theory, macroecological stoichiometry and remotely sensed biogeochemistry. Progress on these emerging frameworks will allow for the integration of ecological stoichiometry and individual space use and fitness.

Infectious disease surveillance is hindered by several factors, including limited infrastructure and geographic isolation of many resource-poor regions. In addition, the complexities of sample acquisition, processing, and analysis, even in developed regions, can be rate limiting. Therefore, new strategies to survey human populations for emerging pathogens are necessary. Xenosurveillance is a method that utilizes mosquitoes as sampling devices to search for genetic signatures of pathogens in vertebrates. Previously we demonstrated that xenosurveillance can detect viral RNA in both laboratory and field settings. However, its ability to detect bacteria and parasites remains to be assessed. Accordingly, we fed Anopheles gambiae mosquitoes blood that contained Trypanosoma brucei gambiense and Bacillus anthracis. In addition, we determined whether two additional emerging viruses, Middle East Respiratory Syndrome Coronavirus and Zika virus could be detected by this method. Pathogen-specific real-time reverse transcription polymerase chain reaction was used to evaluate the sensitivity of xenosurveillance across multiple pathogen taxa and over time. We detected RNA from all pathogens at clinically relevant concentrations from mosquitoes processed up to 1 day postbloodfeeding. These results demonstrate that xenosurveillance may be used as a tool to expand surveillance for viral, parasitic, and bacterial pathogens in resource-limited areas.

Improvements in food hygienic production practices and intervention technologies have reduced the prevalence of pathogen-contaminated carcasses during slaughter. While this unequivocally reduces the risk of foodborne illness, the selection of microbiologic standards based on pathogens for application to raw meat and poultry commodities becomes more burdensome because of the large number of samples required to distinguish between good and poorly performing establishments. This study examines the feasibility of two alternative performance standards based on levels of APC contamination at different locations in the beef slaughter process. An example, based on the Salmonella and E. coli O157:H7 contamination on beef carcasses in the United States provides a case study for the potential effectiveness of the indicator organism-based performance standards. In the example, a performance standard based on the reduction in log10 aerobic plate counts was shown to be superior to a performance standard based on setting a maximum log10 aerobic plate count on finished carcasses.

Arthropod-borne viruses (arboviruses) require efficient replication in taxonomically divergent hosts in order to perpetuate in nature. This review discusses recent advances in our understanding of the phylogenetic position of arthropod-borne viruses relative to insect-specific viruses, which appear to be more common and ecological requirements for successful adoption of the “arbovirus phenotype.” Several molecular and other mechanisms that permit replication in divergent hosts are also discussed.

Background SARS-CoV-2 emerged in 2019 and has become a major global pathogen. Its emergence is notable due to its impacts on individuals residing within long term care facilities (LTCFs) such as rehabilitation centers and nursing homes. LTCF residents tend to possess several risk factors for more severe SARS-CoV-2 outcomes, including advanced age and multiple comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV- 2 deaths in the United States. Methods To assess the prevalence and incidence of SARS-CoV-2 among LTCF workers, determine the extent of asymptomatic SARS-CoV-2 infection, and provide information on the genomic epidemiology of the virus within these unique care settings, we collected nasopharyngeal swabs from workers for 8-11 weeks at six Colorado LTCFs, determined the presence and level of viral RNA and infectious virus within these samples, and sequenced 54 nearly complete genomes. Findings Our data reveal a strikingly high degree of asymptomatic/mildly symptomatic infection, a strong correlation between viral RNA and infectious virus, prolonged infections and persistent RNA in a subset of individuals, and declining incidence over time. Interpretation Our data suggest that asymptomatic SARS-CoV-2 infected individuals contribute to virus persistence and transmission within the workplace, due to high levels of virus. Genetic epidemiology revealed that SARS-CoV-2 likely spreads between staff within an LTCF. Funding Colorado State University Colleges of Health and Human Sciences, Veterinary Medicine and Biomedical Sciences, Natural Sciences, and Walter Scott, Jr. College of Engineering, the Columbine Health Systems Center for Healthy Aging, and the National Institute of Allergy and Infectious Diseases. Research in Context Evidence before this study We searched PubMed and Google Scholar on April 15, 2020 for manuscripts published in 2020 with the key words “SARS-CoV-2 OR COVID-19 AND Long- Term Care Facility AND Surveillance OR Screening. We did not restrict our search to the English language. Our search retrieved two reports of original research. The relevant publications described transmission and course of infection among residents in LTCFs. Of particular relevance was that large quantities of SARS-CoV-2 viral RNA could be detected in asymptomatic, presymptomatic and symptomatic residents, providing early evidence of the heterogeneity of infection characteristics among residents at LTCFs. A significant number of LTCF residents were presymptomatic with symptoms emerging 7 days after initial detection of viral RNA, indicating a longer than expected latency period. Therefore, symptomatic screening for early detection and resultant mitigation response was likely to be ineffective in preventing transmission among residents of LTCFs. There were no reports involving longitudinal surveillance testing of LTCF staff. Added value of this study While prior studies reported results of facility-wide (residents and staff) testing for SARS-CoV-2 and describe transmission dynamics among residents of LTCFs, no prior data was available describing the longitudinal characteristics of SARS-CoV-2 dynamics among staff working at LTCFs during a time period where “shelter-in-place” public guidance was in effect. During this time period, LTCF residents were largely isolated, however staff (those with both direct care and those without direct contact) were permitted to leave and return to work daily. We were therefore interested in this broad staff cohort specifically because they represent a significant and ongoing potential source of transmission within LTCFs. RT-qPCR testing for SARS-CoV-2 was performed weekly on 544 staff in six LTCFs over an 8-11-week period. Symptom data were collected and site-specific prevalence at study onset and incidence rate over time were calculated to explore the influence of identifying and removing asymptomatic SARS-CoV-2-infected individuals from the workplace. Implications of all the available evidence Our results document a surprising degree of asymptomatic/mildly symptomatic infection among apparently healthy staff, and extreme variation in SARS-CoV-2 prevalence and incidence among staff between different facilities. Plaque assay revealed a strong relationship between vRNA and infectious virus in nasopharyngeal swab material, indicating the asymptomatic or mildly symptomatic individuals are infectious. Moreover, phylogenetic analysis of SARS-CoV-2 sequences collected from LTCF staff suggest that the predominant transmission pattern is between staff members within facilities, and that individual unrelated community import events are less common. Finally, decreasing prevalence over time within facilities where longitudinal surveillance testing was performed suggests that identifying and isolating positive staff may serve as part of an effective mitigation program to prevent or curtail transmission among staff within LTCFs.

This chapter addresses the role of subnational government (SNG) borrowing and debt management as the financing conduit for the provision of a nation’s investment in its public infrastructure. It begins with a brief review of recent literature on infrastructure deficit and the link between infrastructure services, economic growth, and development. It then describes intergovernmental organizational and institutional arrangements to promote efficiency in the allocation of scarce resources as well as satisfy the equity goal of matching the cost of using those resources to those who pay for them. Spatial considerations lead to the conclusion that SNGs must play a key role in infrastructure financing. And to play this role, the subnational sector must develop the capacity to take on and manage long-term debt. Having established this normative framework, the discussion turns to organizational and institutional arrangements that must be in place to enable a subnational government to carry out its capital financing and spending role. From there the chapter moves on to address financial risks related to SNG debt finance. Two appendices provide a summary of the intergovernmental fiscal rules for nine East Asian countries and the role of subnational borrowing and debt in China.

BACKGROUND: Ischemic preconditioning (IPC) protects the myocardium against ischemia/reperfusion injury. Evidence suggests that hyperglycemia inhibits IPC-induced cardioprotection. The effects of hyperglycemia initiated during different phases of IPC on myocardial injury were characterized with emphasis on apoptosis and aggregation of polymorphonuclear granulocytes (PMN). METHODS: Male Wistar rats were subjected to 35 min of myocardial ischemia and 2 h of reperfusion. Control animals were not further treated. IPC was induced by three cycles of 3 min ischemia and 5 min of reperfusion before major ischemia. Hyperglycemia (blood glucose more than 22.2 mmol/L) was induced by glucose administration with or without IPC during different phases (trigger- (before ischemia), mediator- (during ischemia), early reperfusion-phase). One additional group received an anti-PMN-antibody before ischemia. Infarct size was quantified by triphenyltetrazolium chloride staining. Cytochrome C release and B-cell lymphoma two (Bcl-2) expression were assessed by western blot analysis. Poly-ADP-Ribose staining and PMN accumulation were quantified with immunohistochemistry and histochemistry. RESULTS: IPC reduced infarct size compared with control. Hyperglycemia completely abolished IPC-induced cardioprotection independent of the time point of initiation. Hyperglycemia before and during major ischemia but without IPC also slightly reduced infarct size. IPC reduced the accumulation of PMNs. This effect was reversed by hyperglycemia during trigger- and mediator-phase but not by hyperglycemia during reperfusion. Hyperglycemia alone had no effect on PMN accumulation. In all treatment groups, signs of myocardial apoptosis were reduced compared with control. IPC alone, combined with hyperglycemia and anti-PMN treatment, reduced apoptosis by a Bcl-2-associated mechanism. Hyperglycemia alone reduced apoptosis by a Bcl-2-independent pathway. CONCLUSION: Hyperglycemia inhibits IPC-induced cardioprotection independent of its onset. Furthermore, hyperglycemia prevents apoptosis and IPC-induced reduction of PMN aggregation.

Cull dairy cattle both on the farm and at slaughter from herds in the states of Idaho, Oregon, and Washington were surveyed for Escherichia coli O157 by culturing fecal swab samples. A total of 205 cull cows from 19 dairy herds were sampled on the farm of origin; 7 (3.4%) tested positive for E. coli O157. A total of 103 cull cows from 15 dairy herds were sampled at slaughter; 4 (3.9%) were positive for E. coli O157. Eighty-nine cull cows were sampled both at the farm and at slaughter; 2 (2.2%) were positive in both locations, 3 (3.3%) only on the farm, and 2 (2.2%) only at the slaughter plant. Seven (7.9%) of the 89 cull cows tracked from farm to slaughter were positive in at least one location. This suggests a higher prevalence of E. coli O157 in cull dairy cattle than previously has been reported to occur in other ages and classes of cattle.

In a 1995 national survey of 937 unpasteurized liquid egg samples collected in breaker plants, 179 of 937 samples (19%) were Salmonella enterica serotype Enteritidis ( S. Enteritidis ) positive. The proportion of unpasteurized liquid egg samples positive for S. Enteritidis was highest in the Northern Region where 106 of 267 samples collected (40%) were S. Enteritidis -positive. These Northern Region results were over three times the S. Enteritidis prevalence detected from the other three regions, whose results ranged between 10% and 12% S. Enteritidis -positive samples. In a 1995 national survey of spent hens at slaughter, 136 of 305 flocks (45%) had at least one S. Enteritidis -positive pooled sample detected. Flock prevalence was highest in the Northern and Central Regions (64% and 40%, respectively); Southeastern and Western Regional flock prevalence levels were much lower (17% and 23%, respectively). A comparison of the 1991 and 1995 unpasteurized liquid egg and spent hen results suggest there has been no decline in S. Enteritidis occurrence in the commercial egg industry between 1991 and 1995. Salmonella Enteritidis phage type 4 was detected in the 1995 surveys of both spent hens and unpasteurized liquid egg but was not found in either survey in 1991. With the exception of one liquid egg sample from the Southeastern Region, S. Enteritidis phage type 4 was found only in the Western Region of the U.S. S. Enteritidis phage type 4 has emerged in the egg industry in the Western U.S. concurrently with an increase in the number of sporadic human phage type 4 isolates in California and Utah.

This cross-sectional study evaluates self-reported behaviors and cases of SARS-CoV-2 infection among essential in-person employees during the first 6 months of the COVID-19 pandemic at a US institution of higher education., Key Points Question What was the prevalence of SARS-CoV-2 infection and associated protective behaviors among essential employees at an institution of higher education during the first 6 months of the COVID-19 pandemic in the US? Findings In this cross-sectional study of 508 essential employees, no cases of SARS-CoV-2 infection were verified by quantitative reverse transcriptase–polymerase chain reaction, and only 2 participants had measurable seroreactive IgG antibodies. High levels of handwashing and mask wearing were reported at work and outside work, and social distancing was reported significantly less often at work than outside work. Meaning The findings suggest that compliance with protective behaviors both at work and outside work may be commensurate with the safe operation of complex work environments during a pandemic., Importance Detailed analysis of infection rates paired with behavioral and employee-reported risk factors is vital to understanding how transmission of SARS-CoV-2 infection may be exacerbated or mitigated in the workplace. Institutions of higher education are heterogeneous work units that supported continued in-person employment during the COVID-19 pandemic, providing a test site for occupational health evaluation. Objective To evaluate the association between self-reported protective behaviors and prevalence of SARS-CoV-2 infection among essential in-person employees during the first 6 months of the COVID-19 pandemic in the US. Design, Setting, and Participants This cross-sectional study was conducted from July 13 to September 2, 2020, at an institution of higher education in Fort Collins, Colorado. Employees 18 years or older without symptoms of COVID-19 who identified as essential in-person workers during the first 6 months of the pandemic were included. Participants completed a survey, and blood and nasal swab samples were collected to assess active SARS-CoV-2 infection via quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and past infection by serologic testing. Exposure Self-reported practice of protective behaviors against COVID-19 according to public health guidelines provided to employees. Main Outcomes and Measures Prevalence of current SARS-CoV-2 infection detected by qRT-PCR or previous SARS-CoV-2 infection detected by an IgG SARS-CoV-2 testing platform. The frequency of protective behavior practices and essential workers’ concerns regarding contracting COVID-19 and exposing others were measured based on survey responses. Results Among 508 participants (305 [60.0%] women, 451 [88.8%] non-Hispanic White individuals; mean [SD] age, 41.1 [12.5] years), there were no qRT-PCR positive test results, and only 2 participants (0.4%) had seroreactive IgG antibodies. Handwashing and mask wearing were reported frequently both at work (480 [94.7%] and 496 [97.8%] participants, respectively) and outside work (465 [91.5%] and 481 [94.7%] participants, respectively). Social distancing was reported less frequently at work (403 [79.5%]) than outside work (465 [91.5%]) (P

Raw meat and poultry contamination frequency demonstrably varies across establishments within an industry. The degree to which more contaminated establishments contribute to the overall production of contaminated products by the entire industry is rarely explored in quantitative microbial risk assessments. We use Lorenz curves and Gini coefficients to describe the degree of concentration of contamination within the industry for several product-pathogen combinations. The analysis is based on beta distributions fit to Salmonella sampling data for comminuted chicken, comminuted turkey, chicken parts, and comminuted beef and beef carcasses, as well as for Campylobacter in chicken and turkey. We also explore empirically-derived Lorenz curves. Lorenz curves for nine product-pathogen pairs suggest the pattern of contamination ranges from highly dispersed across the industry (e.g., Salmonella-comminuted chicken, Gini=0.19) to highly concentrated within a small part of the industry (e.g., Salmonella-beef carcass pre-chill, Gini=0.77). Generally, an inverse relationship between an industry's average contamination frequency and its Gini coefficient is observed across these examples. Also, illustrative empirical Lorenz curves are biased relative to the fitted curves because of a substantial number of empirical results with point estimates of zero. Large Gini coefficients may suggest that risk management should focus on just that part of the industry with high contamination frequencies, while small Gini coefficients might suggest that risk management should be more dispersed and holistic across the industry. We discuss monitoring Gini coefficients across time, as well as other applications of these methods, as useful adjuncts to food safety risk assessments.

We report on findings of the recently completed 2015 Connecticut State and Local Tax Study Panel with a focus on statewide general business taxation. The state's corporate net income (profits) tax ...

West Nile virus remains the most common cause of arboviral encephalitis in North America. Since it was introduced, it has undergone adaptive genetic change as it spread throughout the continent. The WNV transmission cycle is relatively tractable in the laboratory. Thus the virus serves as a convenient model system for studying the population biology of mosquito-borne flaviviruses as they undergo transmission to and from mosquitoes and vertebrates. This review summarizes the current knowledge regarding the population dynamics of this virus within mosquito vectors.

Body-element content was measured for three life stages of wild Atlantic salmon Salmo salar from three distinct Newfoundland populations as individuals crossed between freshwater and marine ecosystems. Life stage explained most of the variation in observed body-element concentration whereas river of capture explained very little variation. Element composition of downstream migrating post-spawn adults (i.e. kelts) and juvenile smolts were similar and the composition of these two life stages strongly differed from adults migrating upstream to spawn. Low variation within life stages and across populations suggests that S. salar may exert rheostatic control of their body-element composition. Additionally, observed differences in trace element concentration between adults and other life stages were probably driven by the high carbon concentration in adults because abundant elements, such as carbon, can strongly influence the observed concentrations of less abundant elements. Thus, understanding variation among individuals in trace elements composition requires the measurement of more abundant elements. Changes in element concentration with ontogeny have important consequences the role of fishes in ecosystem nutrient cycling and should receive further attention.

Our findings do not warrant rejecting the hypothesis that outbreak and sporadic illnesses are similar. Comparing Sporadic and Outbreak Foodborne Illness, Outbreak data have been used to estimate the proportion of illnesses attributable to different foods. Applying outbreak-based attribution estimates to nonoutbreak foodborne illnesses requires an assumption of similar exposure pathways for outbreak and sporadic illnesses. This assumption cannot be tested, but other comparisons can assess its veracity. Our study compares demographic, clinical, temporal, and geographic characteristics of outbreak and sporadic illnesses from Campylobacter, Escherichia coli O157, Listeria, and Salmonella bacteria ascertained by the Foodborne Diseases Active Surveillance Network (FoodNet). Differences among FoodNet sites in outbreak and sporadic illnesses might reflect differences in surveillance practices. For Campylobacter, Listeria, and Escherichia coli O157, outbreak and sporadic illnesses are similar for severity, sex, and age. For Salmonella, outbreak and sporadic illnesses are similar for severity and sex. Nevertheless, the percentage of outbreak illnesses in the youngest age category was lower. Therefore, we do not reject the assumption that outbreak and sporadic illnesses are similar.

Based on the materials of the research carried out in the permanent study areas we analyzed the silvicultural effectiveness of the improvement cutting in the pine forests of Kazakh upland for 58-year period. The improvement cutting in the dry pine forests of Kazakh upland (pine forests of a dead-lichen type) carried out in the 35-year-old pure pine stand even 58 years after can not provide a stock volume similar to that in the control stands where thinning has not been carried out. At the same time the improvement cutting minimize the attrition and increase a mean diameter of a stand, which improves the commercial value of cultivated stands, resistance to adverse natural and anthropogenic factors (recreation, forest fires, etc.), and increases the recreational appeal. Pine plantations of this forest type are characterized by high responsiveness to thinning. Even with a decrease in the relative density in 35-years of age less than 0.5, they maintain stability and by age of 93 (in 58 years after thinning) reproduce the relative density of a normal stand. As thinning improves the sustainability of pine plantations, and all the forests of Kazakh upland belong to the protective purpose forests, we can recommend conducting moderate thinning on stock volume by low thinning in the close 30-40-year-old pine forests of a dead-lichen type.

AIMS Using Bayesian methods that do not require the definition of a gold standard, the diagnostic sensitivity and specificity of a real-time polymerase chain reaction (PCR) assay are compared to those of an enriched culture assay for detection of Campylobacter in enriched comminuted chicken samples. METHODS AND RESULTS Food Safety and Inspection Service comminuted chicken samples were collected from production facilities across the United States. Enriched samples were examined using a commercial real-time PCR kit and plated for culture. Allowing for conditional dependence between these approaches and defining relatively uninformed prior distributions, the 'no gold standard' Bayesian methods generated estimates of the means (95% credible interval) of the posterior distributions for sensitivity and specificity of the PCR as 93% (79, 100%) and 95% (87, 100%) respectively. The estimated sensitivity implies a mean false negative frequency of 7%. The estimated means of the posterior distributions for sensitivity and specificity of the culture assay were 91% (76, 100%) and 96% (88, 100%) respectively. In this case, the mean false negative frequency is 9%. Graphical comparisons of the posterior distributions with their corresponding prior distributions suggested only subtle differences in the sensitivities of both tests, but the posterior distributions for specificities are substantially more certain than the prior distributions. CONCLUSIONS The study suggests that the commercial real-time PCR assay is a more sensitive screening test that would provide timelier negative test results. The modest 1% reduction in specificity of this PCR assay, as compared to an enriched culture assay, is less of a concern for regulatory testing programs if a culture-based confirmatory assay is applied to all presumptive positive samples. SIGNIFICANCE AND IMPACT OF THE STUDY The sensitivity and specificity of a PCR assay and a culture assay for Campylobacter in comminuted poultry produced in the United States were estimated. The PCR assay was shown to be an appropriate alternative screening test.

The genus Flavivirus includes a number of newly recognized viruses that infect and replicate only within mosquitoes. To determine whether insect-specific flaviviruses (ISFs) may infect Culiseta (Cs.) melanura mosquitoes, we screened pools of field-collected mosquitoes for virus infection by RT-PCR targeting conserved regions of the NS5 gene. NS5 nucleotide sequences amplified from Cs. melanura pools were genetically similar to other ISFs and most closely matched Calbertado virus from Culex tarsalis, sharing 68.7% nucleotide and 76.1% amino acid sequence identity. The complete genome of one virus isolate was sequenced to reveal a primary open reading frame (ORF) encoding a viral polyprotein characteristic of the genus Flavivirus. Phylogenetic analysis showed that this virus represents a distinct evolutionary lineage that belongs to the classical ISF group. The virus was detected solely in Cs. melanura pools, occurred in sampled populations from Connecticut, New York, New Hampshire, and Maine, and infected both adult and larval stages of the mosquito. Maximum likelihood estimate infection rates (MLE-IR) were relatively stable in overwintering Cs. melanura larvae collected monthly from November of 2012 through May of 2013 (MLE-IR = 0.7-2.1/100 mosquitoes) and in host-seeking females collected weekly from June through October of 2013 (MLE-IR = 3.8-11.5/100 mosquitoes). Phylogenetic analysis of viral sequences revealed limited genetic variation that lacked obvious geographic structure among strains in the northeastern United States. This new virus is provisionally named Culiseta flavivirus on the basis of its host association with Cs. melanura.

Starting in 2013–2014, the Americas have experienced a massive outbreak of Zika virus (ZIKV) which has now reached at least 49 countries. Although most cases have occurred in South America and the Caribbean, imported and autochthonous cases have occurred in the United States. Aedes aegypti and Aedes albopictus mosquitoes are known vectors of ZIKV. Little is known about the potential for temperate Aedes mosquitoes to transmit ZIKV. Aedes vexans has a worldwide distribution, is highly abundant in particular localities, aggressively bites humans, and is a competent vector of several arboviruses. However, it is not clear whether Ae. vexans mosquitoes are competent to transmit ZIKV. To determine the vector competence of Ae. vexans for ZIKV, wild-caught mosquitoes were exposed to an infectious bloodmeal containing a ZIKV strain isolated during the current outbreak. Approximately 80% of 148 mosquitoes tested became infected by ZIKV, and approximately 5% transmitted infectious virus after 14 days of extrinsic incubation. These results establish that Ae. vexans are competent ZIKV vectors. Their relative importance as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary in ecologically distinct environments.

When interpreting the status of poultry processing establishments subject to performance standards testing, USDA-FSIS employs a round-up rule when fewer than the standard 52 samples per establishment per year are collected. This rule is to ensure that an establishment with fewer than 52 samples is held to a performance standard with a threshold that is not less than that based on the full 52 samples. By estimating population-based sensitivities and specificities for six pathogen reduction performance standards implemented in 2016 (i.e., comminuted chicken, comminuted turkey, and chicken parts standards for both Salmonella and Campylobacter), this study assesses the effect of the round-up rule on correctly classifying commercial poultry establishments as not meeting or meeting the performance standards. Population specificity is greater than 80% for all the performance standards and only modestly affected by reduced sample sizes. Therefore, the round-up rule mitigates against increased misclassification of establishments with low prevalence when sample size is reduced. Population sensitivity is greater than 80% for four of the performance standards but is lower for both comminuted poultry-Campylobacter standards. This sensitivity decreases with reduced sample size and changes sharply as the round up rule is applied. These sensitivity and specificity estimates can serve as benchmarks for future pathogen reduction performance standards, as well as explaining differences with past performance standards.

Evans ML, Kohler AE, Griswold RG, Tardy KA, Eaton KR, Ebel JD. Salmon-mediated nutrient flux in Snake River sockeye salmon nursery lakes: the influence of depressed population size and hatchery supplementation. Lake Reserv Manage. 36:75–86. Since the 1970s, Snake River sockeye salmon (Oncorhynchus nerka) have returned in low numbers to nursery lakes in central Idaho, consequently diminishing marine-derived nutrient subsidies to freshwater spawning and rearing environments. In healthy Pacific salmon populations, returning adults generally import more nutrients than juveniles export, resulting in net positive salmon-mediated nutrient fluxes to freshwater ecosystems. To mitigate for declining sockeye salmon returns, Snake River nursery lakes have been stocked with embryos, with captive-reared juveniles and adults, and fertilized with inorganic nutrients. Here, we examine sockeye salmon-mediated nutrient flux in 3 nursery lakes, Alturas, Pettit, and Redfish, across 20 yr of study (1998–2017). In Redfish Lake, sockeye salmon-mediated nutrient flux was positive in all years when imports from the captive-rearing program were considered in our estimates. Without captive-rearing program inputs, Redfish Lake nutrient flux was positive in only 40% and 45% of years for phosphorus and nitrogen, respectively. In Alturas and Pettit lakes, nutrient export by juveniles exceeded nutrient import by adults in 67% and 56% of years for phosphorus and nitrogen, respectively. Overall, our findings suggest that a sufficient number of adult recruits relative to juvenile migrants, corresponding to a smolt-to-adult return rate > 0.78%, is needed to achieve positive salmon-mediated nutrient flux within lakes and that this will depend on both adequate population productivity and enhanced marine and freshwater habitat connectivity.

Revised Salmonella performance standards for ground chicken and turkey were chosen based on a risk assessment model that predicted a 25% reduction in occurrence of human exposures to Salmonella-contaminated comminuted products following implementation of the standards. To assess the validity of the risk assessment model and its predictions, this study uses a Bayesian framework to compare prior and posterior distributions for model parameters. Prior distributions are informed by 24 months of data collected before the performance standards were implemented. The posterior distributions are generated using a sampling-importance resampling algorithm based on 36 months of data collected after implementing the standards. For the comminuted turkey model parameters, prior and posterior distribution comparisons suggest that the 25% reduction target was accomplished. In contrast, the results for comminuted chicken suggest that the actual reduction in Salmonella was somewhat less than the 25% target. For both commodities, the analysis does not invalidate the risk assessment model, although it does suggest areas for modification in the future.

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.

In urban settings, chikungunya, Zika, and dengue viruses are transmitted by Aedes aegypti mosquitoes. Since these viruses co-circulate in several regions, coinfection in humans and vectors may occur, and human coinfections have been frequently reported. Yet, little is known about the molecular aspects of virus interactions within hosts and how they contribute to arbovirus transmission dynamics. We have previously shown that Aedes aegypti exposed to chikungunya and Zika viruses in the same blood meal can become coinfected and transmit both viruses simultaneously. However, mosquitoes may also become coinfected by multiple, sequential feeds on single infected hosts. Therefore, we tested whether sequential infection with chikungunya and Zika viruses impacts mosquito vector competence. We exposed Ae. aegypti mosquitoes first to one virus and 7 days later to the other virus and compared infection, dissemination, and transmission rates between sequentially and single infected groups. We found that coinfection rates were high after sequential exposure and that mosquitoes were able to co-transmit both viruses. Surprisingly, chikungunya virus coinfection enhanced Zika virus transmission 7 days after the second blood meal. Our data demonstrate heterologous arbovirus synergism within mosquitoes, by unknown mechanisms, leading to enhancement of transmission under certain conditions.

Studies aimed at identifying novel viral sequences or assessing intrahost viral variation require sufficient sequencing coverage to assemble contigs and make accurate variant calling at low frequencies. Many samples come from host tissues where ribosomal RNA represents more than 90% of total RNA preparations, making unbiased sequencing of viral samples inefficient and highly expensive, as many reads will be wasted on cellular RNAs. In the presence of this amount of ribosomal RNA, it is difficult to achieve sufficient sequencing depth to perform analyses such as variant calling, haplotype prediction, virus population analyses, virus discovery or transcriptomic profiling. Many methods for depleting unwanted RNA or enriching RNA of interest have been devised, including poly-A selection, RNase H based specific depletion, duplex-specific nuclease treatment and hybrid capture selection, among others. Although these methods can be efficient, they either cannot be used for some viruses (i.e. non-polyadenylated viruses), have been optimized for use in a single species, or have the potential to introduce bias. In this study, we describe a novel approach that uses an RNaseH possessing reverse transcriptase coupled with selective probes for ribosomal RNA designed to work broadly for three medically relevant mosquito genera;Aedes,Anopheles,andCulex.We demonstrate significant depletion of rRNA using multiple assessment techniques from a variety of sample types, including whole mosquitoes and mosquito midgut contents from FTA cards. To demonstrate the utility of our approach, we describe novel insect-specific virus genomes from numerous species of field collected mosquitoes that underwent rRNA depletion, thereby facilitating their detection. The protocol is straightforward, relatively low-cost and requires only common laboratory reagents and the design of several small oligonucleotides specific to the species of interest. This approach can be adapted for use with other organisms with relative ease, thus potentially aiding virus population genetics analyses, virus discovery and transcriptomic profiling in both laboratory and field samples.

Buffered peptone water is the rinsate commonly used for chicken rinse sampling. A new formulation of buffered peptone water was developed to address concerns about the transfer of antimicrobials, used during poultry slaughter and processing, into the rinsate. This new formulation contains additives to neutralize the antimicrobials, and this neutralizing buffered peptone water replaced the original formulation for all chicken carcass and chicken part sampling programs run by the Food Safety and Inspection Service beginning in July 2016. Our goal was to determine whether the change in rinsate resulted in significant differences in the observed proportion of positive chicken rinse samples for both Salmonella and Campylobacter. This assessment compared sampling results for the 12-month periods before and after implementation. The proportion of carcass samples that tested positive for Salmonella increased from approximately 0.02 to almost 0.06. Concurrently, the proportion of chicken part samples that tested for Campylobacter decreased from 0.15 to 0.04. There were no significant differences associated with neutralizing buffered peptone water for the other two product-pathogen pairs. Further analysis of the effect of the new rinsate on corporations that operate multiple establishments demonstrated that changes in the percent positive rates differed across the corporations, with some corporations being unaffected, while others saw all of the establishments operated by the corporation move from passing to failing the performance standard and vice versa. The results validated earlier concerns that antimicrobial contamination of rinse samples was causing false-negative Salmonella testing results for chicken carcasses. The results also indicate that additional development work may still be required before the rinsate is sufficiently robust for its use in Campylobacter testing.

Congenital Zika virus (ZIKV) infection was first linked to birth defects during the American outbreak 1–3. It has been proposed that mutations unique to the Asian/American-genotype explain, at least in part, the ability of Asian/American ZIKV to cause congenital Zika syndrome (CZS) 4,5. Recent studies identified mutations in ZIKV infecting humans that arose coincident with the outbreak in French Polynesia and were stably maintained during subsequent spread to the Americas 5. Here we show that African ZIKV can infect and harm fetuses and that the S139N mutation that has been associated with the American outbreak is not essential for fetal harm. Our findings, in a vertical transmission mouse model, suggest that ZIKV will remain a threat to pregnant women for the foreseeable future, including in Africa, southeast Asia, and the Americas. Additional research is needed to better understand the risks associated with ZIKV infection during pregnancy, both in areas where the virus is newly endemic and where it has been circulating for decades.

Ixodes scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic, but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled, and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence.

Zika virus (ZIKV) has challenged the assumed knowledge regarding the pathobiology of flaviviruses. Despite causing sporadic and mild disease in the 50 years since its discovery, Zika virus has now caused multiple outbreaks in dozens of countries worldwide. Moreover, the disease severity in recent outbreaks, with neurological disease in adult and devastating congenital malformations in fetuses, was not previously seen. One hypothesis is that the virus has acquired mutations that have increased its virulence. Indeed, mutations in other arboviruses, such as West Nile virus (WNV), chikungunya virus (CHIKV), and Venezuelan equine encephalitis virus (VEEV), have enhanced outbreaks. This possibility, as well as alternative hypotheses, are explored here.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. We developed CATCH (Compact Aggregation of Targets for Comprehensive Hybridization), a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs compact probe sets that achieve full coverage of known sequence diversity and that scale well with this diversity. To illustrate applications of CATCH, we focused on capturing viral genomes. We designed, synthesized, and validated multiple probe sets, including one that targets whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriched unique viral content on average 18× and allowed us to assemble genomes that we could not otherwise recover, while accurately preserving within-sample diversity. We used this approach to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of viral infections in samples with unknown content. Together, this work demonstrates a path toward more sensitive, cost-effective metagenomic sequencing.

Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus. Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3′ UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes.

Food-safety regulatory agencies are often tasked with oversight of a broad range of food commodities. For these agencies to regulate multiple commodities effectively, they need to develop policies and allocate resources that consider the varying magnitudes of the risk of illness that each of the commodities poses to the broad population of consumers. Process modeling is used in risk assessment to estimate the likelihood of illness by modeling contamination of raw foods, the microbial dynamics of pathogens between production and consumption, and dose–response relationships for the pathogen to estimate the risk and total number of illnesses for a specific commodity. Nevertheless, these models are usually unique to each commodity and constructed using different models and data sources, which can produce estimates that are difficult to compare. An alternative approach is presented that stems primarily from public health data. It uses simple methods to estimate various risk metrics simultaneously for multiple pathogens and commodities. This alternative approach is used to compare multiple risk metrics for beef, lamb, pork, and poultry for both Salmonella and Escherichia coli O157:H7. The implications of the different risk metrics are discussed with respect to current regulatory efforts in the United States.

SUMMARYHuman illness attribution is recognized as an important metric for prioritizing and informing food-safety decisions and for monitoring progress towards long-term food-safety goals. Inferences regarding the proportion of illnesses attributed to a specific commodity class are often based on analyses of datasets describing the number of outbreaks in a given year or combination of years. In many countries, the total number of pathogen-related outbreaks reported nationwide for an implicated food source is often fewer than 50 instances in a given year and the number of years for which data are available can be fewer than 10. Therefore, a high degree of uncertainty is associated with the estimated fraction of pathogen-related outbreaks attributed to a general food commodity. Although it is possible to make inferences using only data from the most recent year, this type of estimation strategy ignores the data collected in previous years. Thus, a strong argument exists for an estimator that could ‘borrow strength’ from data collected in the previous years by combining the current data with the data from previous years. While many estimators exist for combining multiple years of data, most either require more data than is currently available or lack an objective and biologically plausible theoretical basis. This study introduces an estimation strategy that progressively reduces the influence of data collected in past years in accordance with the degree of departure from a Poisson process. The methodology is applied to the estimation of the attribution fraction forSalmonellaandEscherichia coliO157:H7 for common food commodities and the estimates are compared against two alternative estimators.

Pork and poultry products have been implicated in a substantial proportion of microbial foodborne illnesses such as salmonellosis and campylobacteriosis. Understanding the similarities and differences between the effectiveness of interventions applied during slaughter to the two commodities can help identify opportunities to reduce the risk of foodborne illness. This study uses data from two national surveys and censored data techniques to assess the concentrations of microbial contamination on broiler chickens and market hogs at two points in the slaughter process. Statistical distributions are fitted to estimate the average concentrations of microbial contamination, the variability in these concentrations, and the average reduction in contamination between the two locations for pork and chicken produced in the United States. The concentrations of Salmonella and generic Escherichia coli (GEC) are estimated for hog carcasses; the concentrations of Salmonella , GEC, and Campylobacter are estimated for chicken carcasses. These datasets are also used to study the magnitude of seasonal changes in concentrations of microbial contamination and to demonstrate the potential utility of using the reduction in the average aerobic plate count between the pre-evisceration and post-chill stages of the slaughter process as an indicator of the corresponding reduction in other species of bacteria. The study finds interventions for hog carcasses achieve much larger reductions in contamination than those applied to chicken. However, the variability in the concentration of Salmonella contamination on pork is significantly higher, suggesting an opportunity to decrease risk by ensuring more consistent reductions across the industry. The study also finds, for both commodities, that the log 10 reductions in the concentrations of GEC are similar to the log 10 reductions in Salmonella , while the log reduction in Campylobacter on chicken was substantially greater than for both GEC and Salmonella . Additional analyses demonstrate modest seasonal increases in contamination only on hog carcasses as well as demonstrating that reductions in APC are indicative of reductions in all other organisms, suggesting its potential as an effective indicator organism.

To test the hypothesis that RNA interference (RNAi) imposes diversifying selection on RNA virus genomes, we quantified West Nile virus (WNV) quasispecies diversity after passage in Drosophila cells in which RNAi was left intact, depleted, or stimulated against WNV. As predicted, WNV diversity was significantly lower in RNAi-depleted cells and significantly greater in RNAi-stimulated cells relative to that in controls. These findings reveal that an innate immune defense can shape viral population structure.

Arboviruses such as West Nile, Zika, chikungunya, dengue, and yellow fever viruses have become highly significant global pathogens through unexpected, explosive outbreaks. While the rapid progression and frequency of recent arbovirus outbreaks is associated with long-term changes in human behavior (globalization, urbanization, climate change), there are direct mosquito–virus interactions which drive shifts in host range and alter virus transmission. This review summarizes how virus–mosquito interactions are critical for these viruses to become global pathogens at molecular, physiological, evolutionary, and epidemiological scales. Integrated proactive approaches are required in order to effectively manage the emergence of mosquito-borne arboviruses, which appears likely to continue into the indefinite future.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a “synthetic swarm” whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics., Author summary Understanding the complex dynamics of Zika virus (ZIKV) infection during pregnancy and during transmission to and from vertebrate host and mosquito vector is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and reservoir establishment. We sought to develop a virus model system for use in nonhuman primates and mosquitoes that allows for the genetic discrimination of molecularly cloned viruses. This “synthetic swarm” of viruses incorporates a molecular barcode that allows for tracking and monitoring individual viral lineages during infection. Here we infected rhesus macaques with this virus to study the dynamics of ZIKV infection in nonhuman primates as well as during mosquito infection/transmission. We found that the proportions of individual barcoded viruses remained relatively stable during acute infection in pregnant and nonpregnant animals. However, in a pregnant animal, the complexity of the virus population declined precipitously 8 days following infection, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a “synthetic swarm” whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcode clonotypes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defectsin vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 64 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques, and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over timein vivoto understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.Author summaryUnderstanding the complex dynamics of Zika virus (ZIKV) infection during pregnancy and during transmission to and from vertebrate host and mosquito vector is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and reservoir establishment. We sought to develop a virus model system for use in nonhuman primates and mosquitoes that allows for the genetic discrimination of molecularly cloned viruses. This “synthetic swarm” of viruses incorporates a molecular barcode that allows for tracking and monitoring individual viral lineages during infection. Here we infected rhesus macaques with this virus to study the dynamics of ZIKV infection in nonhuman primates as well as during mosquito infection/transmission. We found that the proportions of individual barcoded viruses remained relatively stable during acute infection in pregnant and nonpregnant animals. However, in a pregnant animal, the complexity of the virus population declined precipitously 8 days following infection, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques, and remained low for the subsequent duration of viremia.

Advances in microbiological testing methods have led to faster and less expensive assays. Given these advances, it is logical to employ these assays for use in the sampling plan of an existing microbiological criterion. A change in the performance characteristics of the assay can affect the intended effect of the microbiological criterion. This study describes a method for updating a 2-class attributes sampling plan to account for the different test sensitivity and specificity of a new assay and provides an example based on the replacement of a culture-based assay with a real-time polymerase chain reaction assay.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

Zika virus (ZIKV; Genus Flavivirus, Family Flaviviridae) has recently emerged in Asia and the Americas to cause large outbreaks of human disease. The outbreak has been characterized by high attack rates, birth defects in infants and severe neurological complications in adults. ZIKV is transmitted to humans by Aedes mosquitoes, but recent evidence implicates sexual transmission as playing an important role as well. This review highlights the transmission of ZIKV in humans, with a focus on both mosquito and sexually-transmitted routes and their outcomes. We also discuss critical directions for future research.

Background ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. Methodology/Principal findings Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59—Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. Conclusions/Significance Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8–51% in Ae. aegypti and from 2–26% in Ae. albopictus., Author summary Aedes aegypti is an efficient vector for arboviruses which is partially determined by its vector competence (VC) status, a highly variable trait. Based on previous reports, the VC of New World mosquitoes is limited for ZIKV. However, a VC assessment from additional geographical sources was lacking. Therefore, we evaluated the VC for ZIKV using recently colonized mosquitoes from Mexico. Aedes aegypti and Ae. albopictus were highly susceptible to ZIKV infection but varied greatly in their transmission rates. As with previous studies of other flaviviruses; VC for ZIKV was highly variable in both Ae. aegypti and Ae. albopictus but was greatest in Ae. aegypti, supporting its role as the main ZIKV vector.