Surveillance and Genetic Analysis of Jamestown Canyon Virus in New York State: 2001-2022.

Jamestown Canyon virus (JCV) (Peribunyavirdae; Orthobunyavirus) is a mosquito-borne pathogen endemic to North America. The genome is composed of three segmented negative-sense RNA fragments designated as small, medium, and large. Jamestown Canyon virus is an emerging threat to public health, and infection in humans can cause severe neurological diseases, including encephalitis and meningitis. We report JCV mosquito surveillance data from 2001 to 2022 in New York state. Jamestown Canyon virus was detected in 12 mosquito species, with the greatest prevalence in Aedes canadensis and Anopheles punctipennis. Detection fluctuated annually, with the highest levels recorded in 2020. Overall, JCV infection rates were significantly greater from 2012 to 2022 compared with 2001 to 2011. Full-genome sequencing and phylogenetic analysis were also performed with representative JCV isolates collected from 2003 to 2022. These data demonstrated the circulation of numerous genetic variants, broad geographic separation, and the first identification of lineage B JCV in New York state in 2022.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Bourbon Virus Transmission, New York, USA.

In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

Adaptive evolution of West Nile virus facilitated increased transmissibility and prevalence in New York State.

West Nile virus (WNV; Flavivirus, Flaviviridae) was introduced to New York State (NYS) in 1999 and rapidly expanded its range through the continental United States (US). Apart from the displacement of the introductory NY99 genotype with the WN02 genotype, there has been little evidence of adaptive evolution of WNV in the US. WNV NY10, characterized by shared amino acid substitutions R1331K and I2513M, emerged in 2010 coincident with increased WNV cases in humans and prevalence in mosquitoes. Previous studies demonstrated an increase in frequency of NY10 strains in NYS and evidence of positive selection. Here, we present updated surveillance and sequencing data for WNV in NYS and investigate if NY10 genotype strains are associated with phenotypic change consistent with an adaptive advantage. Results confirm a significant increase in prevalence in mosquitoes though 2018, and updated sequencing demonstrates a continued dominance of NY10. We evaluated NY10 strains in Culex pipiens mosquitoes to assess vector competence and found that the NY10 genotype is associated with both increased infectivity and transmissibility. Experimental infection of American robins (Turdus migratorius) was additionally completed to assess viremia kinetics of NY10 relative to WN02. Modelling the increased infectivity and transmissibility of the NY10 strains together with strain-specific viremia demonstrates a mechanistic basis for selection that has likely contributed to the increased prevalence of WNV in NYS.

Aedes Albopictus and Cache Valley virus: a new threat for virus transmission in New York State.

We report surveillance results of Cache Valley virus (CVV; Peribunyaviridae, Orthobunyavirus) from 2017 to 2020 in New York State (NYS). Infection rates were calculated using the maximum likelihood estimation (MLE) method by year, region, and mosquito species. The highest infection rates were identified among Anopheles spp. mosquitoes and we detected the virus in Aedes albopictus for the first time in NYS. Based on our previous Anopheles quadrimaculatus vector competence results for nine CVV strains, we selected among them three stains for further characterization. These include two CVV reassortants (PA and 15041084) and one CVV lineage 2 strain (Hu-2011). We analyzed full genomes, compared in vitro growth kinetics and assessed vector competence of Aedes albopictus. Sequence analysis of the two reassortant strains (PA and 15041084) revealed 0.3%, 0.4%, and 0.3% divergence; and 1, 10, and 6 amino acid differences for the S, M, and L segments, respectively. We additionally found that the PA strain was attenuated in vertebrate (Vero) and mosquito (C6/36) cell culture. Furthemore, Ae. albopictus mosquitoes are competent vectors for CVV Hu-2011 (16.7-62.1% transmission rates) and CVV 15041084 (27.3-48.0% transmission rates), but not for the human reassortant (PA) isolate, which did not disseminate from the mosquito midgut. Together, our results demonstrate significant phenotypic variability among strains and highlight the capacity for Ae. albopictus to act as a vector of CVV.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Spatial and temporal expansions of Eastern equine encephalitis virus and phylogenetic groups isolated from mosquitoes and mammalian cases in New York State from 2013 to 2019.

Surveillance for the emerging infectious disease Eastern equine encephalitis, and its causative virus in mosquitoes, continued within New York State from 2013 to 2019. There were increases in geographic area and number of consecutive years, with cases in four mammalian species, and virus in 11 mosquito species. The first cases in a goat and in an emu were reported. The first detection of virus in Aedes cinereus was reported. Virus in phylogenetic group NY4 was isolated from a horse and from mosquitoes 6 kilometers and 13 days apart in 2013. Phylogenetic groups NY4 and NY5 were found 15 days apart in two towns 280 kilometers distant in 2013. Within four adjacent counties there was a pattern of overlap, where four had NY5, two adjacent counties had NY6, two adjacent counties had NY7, and one county had NY5, NY6, and NY7, reducible to a Euler diagram. Virus in phylogenetic group NY5, found within an 11-kilometer wide area in New York State, was related to FL4 found in Florida 1,398 kilometers distant. This was consistent with a phylogenetic group originating in Florida, then being moved to a specific location in New York State, by migratory birds in consecutive years 2013 and 2014.

Integrated Arbovirus Surveillance Improves the Detection Onset of Zika Virus in Panama.

We tested 700 serum samples collected throughout Panama from 2015 to 2016 for detecting antibodies and RNA of arboviruses. In convalescent specimens, microsphere immunoassay detected an antibody prevalence of 59.3% for dengue virus (DENV) and 30.3% for Zika virus (ZIKV), which included samples that were collected before the Panamanian surveillance system reported the first case of Zika in the country. For acute sera, the most common arbovirus was DENV with 18 positive samples (6%), followed by four (1.3%) of ZIKV and one (0.6%) of chikungunya virus (CHIKV). Our results indicate a change in the chronology of when ZIKV was first detected in Panama and stress the importance of integrating various approaches to enable improved surveillance of both endemic and emerging arboviruses.

Erratum: Evolutionary dynamics and molecular epidemiology of West Nile virus in New York State: 1999-2015.

[This corrects the article DOI: 10.1093/ve/vez020.].

Evolutionary dynamics and molecular epidemiology of West Nile virus in New York State: 1999-2015.

Following its introduction into New York State (NYS) in 1999, West Nile virus (WNV; Flavivirus, Flaviviridae) underwent a rapid expansion throughout the USA and into Canada and Latin America. WNV has been characterized as being evolutionarily stable, with weak geographic structure, a dominance of purifying selection and limited adaptive change. We analyzed all available full-genome WNV sequences, focusing on the 543 available sequences from NYS, which included 495 newly sequenced 2000-15 isolates. In addition, we analyzed deep-sequencing data from 317 of these isolates. While our data are generally in agreement with the limited pace of evolutionary change and broad geographic and temporal mixing identified in other studies, we have identified some important exceptions. Most notably, there are 14 codons which demonstrated evidence of positive selection as determined by multiple models, including some positions with evidence of selection in NYS exclusively. Coincident with increased WNV activity, genotypes possessing one or more of these mutations, designated NY01, NY07, and NY10, have increased in prevalence in recent years and displaced historic strains. In addition, we have found a geographical bias with many of these mutations, which suggests selective pressures and adaptations could be regional. Lastly, our deep-sequencing data suggest both increased overall diversity in avian tissue isolates relative to mosquito isolates and multiple non-synonymous minority variants that are both host-specific and retained over time and space. Together, these data provide novel insight into the evolutionary pressures on WNV and the need for continued genetic surveillance and characterization of emergent strains.

Influence of Forest Disturbance on La Crosse Virus Risk in Southwestern Virginia.

Forest disturbance effects on La Crosse virus (LACV) are currently unknown. We determined the abundance of three LACV accessory vectors (Aedes albopictus, Ae. canadensis, and Ae. vexans) and the primary amplifying host (Eastern chipmunk; Tamias striatus), and tested for LACV prevalence in both vectors and chipmunks, across a gradient of experimental forest disturbance treatments in southwest Virginia. Forest disturbance significantly affected the abundance of LACV accessory vectors, with a higher abundance on disturbed sites for Ae. canadensis and Ae. vexans. However, there was no significant disturbance effect on chipmunk abundance. Forest disturbance significantly affected LACV prevalence in mosquito vectors, with most (80%) detections on unlogged control sites, which past work showed harbor the highest abundance of the two most common LACV vectors (the primary vector Aedes triseriatus, and Ae. japonicus). Interestingly, LACV nucleic acid was only detected in Ae. japonicus and Culex pipiens/restuans, with no detections in the primary vector, Ae. triseriatus. In contrast to the vector results, antibodies were only found in chipmunks on logged sites, but this result was not statistically significant. Overall, our results suggest that human LACV risk should generally decline with logging, and reveal the potential importance of accessory vectors in LACV maintenance in Appalachian forests.

Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence.

In the Western Hemisphere, Zika virus is thought to be transmitted primarily by Aedes aegypti mosquitoes. To determine the extent to which Ae. albopictus mosquitoes from the United States are capable of transmitting Zika virus and the influence of virus dose, virus strain, and mosquito species on vector competence, we evaluated multiple doses of representative Zika virus strains in Ae. aegypti and Ae. albopictus mosquitoes. Virus preparation (fresh vs. frozen) significantly affected virus infectivity in mosquitoes. We calculated 50% infectious doses to be 6.1-7.5 log 10 PFU/mL; minimum infective dose was 4.2 log 10 PFU/mL. Ae. albopictus mosquitoes were more susceptible to infection than Ae. aegypti mosquitoes, but transmission efficiency was higher for Ae. aegypti mosquitoes, indicating a transmission barrier in Ae. albopictus mosquitoes. Results suggest that, although Zika virus transmission is relatively inefficient overall and dependent on virus strain and mosquito species, Ae. albopictus mosquitoes could become major vectors in the Americas.

Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens.

Complex interactions between microbial residents of mosquitoes and arboviruses are likely to influence many aspects of vectorial capacity and could potentially have profound effects on patterns of arbovirus transmission. Such interactions have not been well studied for West Nile virus (WNV; Flaviviridae, Flavivirus) and Culex spp. mosquitoes. We utilized next-generation sequencing of 16S ribosomal RNA bacterial genes derived from Culex pipiens Linnaeus following WNV exposure and/or infection and compared bacterial populations and broad immune responses to unexposed mosquitoes. Our results demonstrate that WNV infection increases the diversity of bacterial populations and is associated with up-regulation of classical invertebrate immune pathways including RNA interference (RNAi), Toll, and Jak-STAT (Janus kinase-Signal Transducer and Activator of Transcription). In addition, WNV exposure alone, without the establishment of infection, results in similar alterations to microbial and immune signatures, although to a lesser extent. Multiple bacterial genera were found in greater abundance inWNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia.

Effectiveness of Evidence-based Pneumonia CPOE Order Sets Measured by Health Outcomes.

OBJECTIVE: Evidence-based sets of medical orders for the treatment of patients with common conditions have the potential to induce greater efficiency and convenience across the system, along with more consistent health outcomes. Despite ongoing utilization of order sets, quantitative evidence of their effectiveness is lacking. In this study, conducted at Advocate Health Care in Illinois, we quantitatively analyzed the benefits of community acquired pneumonia order sets as measured by mortality, readmission, and length of stay (LOS) outcomes. METHODS: In this study, we examined five years (2007-2011) of computerized physician order entry (CPOE) data from two city and two suburban community care hospitals. Mortality and readmissions benefits were analyzed by comparing "order set" and "no order set" groups of adult patients using logistic regression, Pearson's chi-squared, and Fisher's exact methods. LOS was calculated by applying one-way ANOVA and the Mann-Whitney U test, supplemented by analysis of comorbidity via the Charlson Comorbidity Index. RESULTS: The results indicate that patient treatment orders placed via electronic sets were effective in reducing mortality [OR=1.787; 95% CF 1.170-2.730; P=.061], readmissions [OR=1.362; 95% CF 1.015-1.827; P=.039], and LOS [F (1,5087)=6.885, P=.009, 4.79 days (no order set group) vs. 4.32 days (order set group)]. CONCLUSION: Evidence-based ordering practices have the potential to improve pneumonia outcomes through reduction of mortality, hospital readmissions, and cost of care. However, the practice must be part of a larger strategic effort to reduce variability in patient care processes. Further experimental and/or observational studies are required to reduce the barriers to retrospective patient care analyses.

La Crosse Virus in Aedes japonicus japonicus mosquitoes in the Appalachian Region, United States.

La Crosse virus (LACV), a leading cause of arboviral encephalitis in children in the United States, is emerging in Appalachia. For local arboviral surveillance, mosquitoes were tested. LACV RNA was detected and isolated from Aedes japonicus mosquitoes. These invasive mosquitoes may significantly affect LACV range expansion and dynamics.

Effectiveness of Evidence-Based Congestive Heart Failure (CHF) CPOE Order Sets Measured by Health Outcomes.

OBJECTIVE: Evidence-based order sets for treatment of patients with common conditions promise ordering efficiency and more consistent health outcomes. Despite ongoing utilization of order sets, quantitative evidence of their effectiveness is lacking. This study quantitatively analyzed benefits of CHF order sets as measured by mortality, readmission, and length of stay (LOS) outcomes. METHODS: Mortality and readmissions were analyzed by comparing "order set" and "free text" groups of adult patients using logistic regression, Pearson chi-squared, and Fisher's exact methods. LOS was calculated by applying One-Way ANOVA and Mann-Whitney tests, supplemented by comorbidity analysis via Charlson Comorbidity Index. RESULTS: CHF orders placed via sets were effective in reducing mortality [OR=1.818;95% CF 1.039-3.181;p=0.034] and LOS [F(1,10938)=8.352,p=0.013,4.75 days ("free text" group) vs. 5.46 days ("order set" group)], while readmission outcome was not significant [OR=0.913;95% CF 0.734-1.137;p=0.417]. CONCLUSION: Evidence-based medication ordering practices to treat CHF have potential to reduce mortality and LOS, without effect on readmissions.

Quadraplex qRT-PCR assay for the simultaneous detection of Eastern equine encephalitis virus and West Nile virus.

In order to increase testing throughput and reduce cost, we developed a multiplex real-time assay that identifies both Eastern equine encephalitis virus and West Nile virus. The assay allows for the screening for the presence of both the nonstructural and envelope genes of both viruses simultaneously allowing for confirmatory testing to be done in a single assay. We utilized newly designed primers and probes, each labeled with a unique fluorescent label allowing for differentiation using an ABI 7500 real-time PCR machine. The use of Quanta Biosciences qScript XLT One-Step RT-qPCR® Toughmix allowed for a quadraplex assay without loss of sensitivity when compared to the previously run singleplex reaction as seen with viral RNA PFU control dilution series. There was no cross reactivity between the viruses within the reaction, and upon utilization of the assay during surveillance, there was no cross reactivity with other historically encountered arthropod-borne viruses. The results from the quantitative Reverse Transcriptase - Polymerase Chain Reaction were comparable to those achieved by cell culture which was performed on a subset of the field mosquito pools screened during the 2012 surveillance season. The multiplex assay resulted in savings in both time and resources for the lab and faster turn-around of results.

Importance of srtA and srtB for growth of Bacillus anthracis in macrophages.

We examined the effect of mutation of two sortase genes of Bacillus anthracis, srtA and srtB, on the ability of the bacterium to grow in J774A.1 cells, a mouse macrophage-like cell line. While disruption of either srtA or srtB had no effect on the ability of the bacteria to grow in rich culture media, mutations in each of these genes dramatically attenuated growth of the bacterium in J774A.1 cells. Complementation of the mutation restored the ability of bacteria to grow in the cells. Since the initial events in inhalation anthrax are believed to be uptake of B. anthracis spores by alveolar macrophages followed by germination of the spores and growth of the bacteria within the macrophages, these results suggest that two sortases of B. anthracis may be critical in the early stages of inhalation anthrax.

Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome.

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for evasion of endocytic fusion and for activation of caspase-3 during early stages of infection of macrophages, but the mechanisms of manipulating these host cell processes are not known. Here, we show that caspase-3 activation by L. pneumophila is independent of all the known apoptotic pathways that converge on the activation of caspase-3. The cytoplasmic proteins IcmS, IcmR and IcmQ, which are involved in secretion of Dot/Icm effectors, are required for caspase-3 activation. Pretreatment of U937 macrophages and human peripheral blood monocytes (hPBM) with the capase-3 inhibitor (DEVD-fmk) or the paninhibitor of caspases (Z-VAD-fmk) before infection blocks intracellular replication of L. pneumophila in a dose-dependent manner. Inhibition of caspase-3 results in co-localization of the L. pneumophila-containing phagosome (LCP) with the late endosomal/lysosomal marker Lamp-2, and the LCP contains lysosomal enzymes, similar to the dotA mutant, which is defective in caspase-3 activation. However, activation of caspase-3 before infection does not rescue the replication defect of the dotA mutant. Interestingly, inhibition of caspase-3 after a 15 or 30 min infection period by the parental strain has no detectable effect on the formation of a replicative niche. The Dot/Icm-mediated activation of caspase-3 by L. pneumophila specifically cleaves, in a dose- and time-dependent manner, the Rab5 effector Rabaptin-5, which maintains Rab5-GTP on the endosomal membrane. In addition, PI3 kinase, which is a crucial effector of Rab5 downstream of Rababptin-5, is not required for intracellular replication. Using single-cell analysis, we show that apoptosis is not evident in the infected cell until bacterial replication results in > 20 bacteria per cell. We conclude that activation of caspase-3 by the Dot/Icm virulence system of L. pneumophila is essential for halting biogenesis of the LCP through the endosomal/lysosomal pathway, and that this is associated with the cleavage of Rabpatin-5.