Advances in understanding influenza A virus infection in swine: From diagnostics to basic science

Influenza A virus (IAV) is a member of the family Orthomyxoviridae and is considered a major pathogen for a number of species including humans, swine, and avian species. Historically, the potential of avian and swine influenza A viruses, either through direct infection by intact virus or through incorporation of genetic information into human isolates, have presented major zoonotic risks to human health risks with epidemic and pandemic potential (98). Prior to the 2009 pandemic, experts were concerned that high pathogenic avian IAV, particularly H5N1, was going to become established in the human population and that such establishment would lead to a pandemic crisis due to the lack of human population immunity to the avian IAV (24, 96). One hypothesized mechanism by which avian IAV could establish itself in the human population is through adaptation in an intermediate host, such as a pig (50). Because such a possibility may exist, determination of the prevalence of H5N1 infection in the swine populations in regions endemic for highly pathogenic H5N1 viruses is highly desirable. Though the inherent pathogenicity of an intact wildtype virus for its natural host is evident in field or experimental infections, incorporation of the genetic factor(s) responsible for pathogenicity from one virus to another may result in disease that is just as severe. One such pathogenicity factor is the PB1-F2 protein found in a secondary open reading frame of the PB1 mRNA. The PB1-F2 protein is relatively small when compared to other influenza proteins but is associated with a number of pathogenicity factors, including apoptosis, immunopathology, and secondary bacterial infection (23, 63, 105). Though a fair amount of work has been done to understand PB1-F2 in human and avian isolates, currently little is known about the function and expression levels of PB1-F2 during infection with swine influenza virus isolates. Given the zoonotic potential of swine influenza viruses, it is important to understand the effect that PB1-F2 could have on both human and animal health

Population Characteristics and Habitat Use by the Recently Introduced Asiatic Clam (Corbicula fluminea) in Lake Whatcom, Washington

The Asiatic Clam (Corbicula fluminea) was found in Lake Whatcom in 2011. This exotic clam is common throughout North America and is spread between watersheds by infested boats, fishing activities, as well as passively by waterfowl. Corbicula fluminea is a well documented invasive species that survives in many environments and exhibits an rselected life history which can lead to potentially rapid population growth via a clonal reproductive ability typical among invasive bivalves and members of the family Corbiculidae. There are more reproductive strategies in Corbiculidae than any other freshwater bivalve. This rapid growth of a single organism and its associated consumption and excretions can lead to undesired changes in an aquatic ecosystem. Studies have shown a drop in species richness, alterations to algal communities and their availability to other organisms, and water quality changes associated with burrowing, shell accumulation, and clam decomposition. My research included an assessment of the growth of representative Lake Whatcom clam populations during 2012 and 2013 using shellfish surveying methods that have been applied to the marine intertidal environment. Surveying was based on multiple transects with randomly sampled 0.25-square meter quadrats. Three sites were identified that had populations of the clam and were accessible for surveys. These sites were Bloedel Donovan Park in the City of Bellingham, Lakewood, a facility run by Western Washington University, and a small park beach within the community of Sudden Valley. Surveys showed sample areas with 200 or more individual clams per square meter at all three sites. Studies state this density to be indicative of a self-sustaining population for C. fluminea. Some sites exhibited an increase in biomass and size from 2012 to 2013. All sites showed significant changes among some size classes that suggest growth. The sand and fine sediment substrate of the Sudden Valley site hosted significant density increases and biomass increases from 2012 to 2013. The harder rocky substrate of Lakewood hosted multiple size classes but did not show evidence of growth. Bloedel Donovan Park differed from the other sites in that it had a small size class in 2013 that was not present in 2012 suggesting a new generation of clams had reseeded the habitat. The overall environment within Lake Whatcom does not appear to be conducive to extended periods of reproduction based on the presence of distinct size classes. Distinct size classes are representative of specific reproductive windows during the year made available during the warmer months of summer. Density and biomass changed with depth within the nearshore shallows suggesting that the cooler deeper waters of the lake are not as suitable to the clam as the warmer, shallower areas within the littoral zone. Another explanation is less phytoplankton availability due to light limitations imposed by depth. Corbicula fluminea appears to be reproducing to varying degrees at all three sites in this study, and it will likely continue to spread to suitable habitat within Lake Whatcom. Typical impacts associated with the clam should be expected. These include changes in species richness, especially changes in native filter feeder concentration as well as changes to phytoplankton density, and alterations to the seston nutrient load because of burrowing and biological functions associated with C. flumine

Issues encountered in development of enzymelinked immunosorbent assay for use in detecting \u3ci\u3eInfluenza A virus\u3c/i\u3e subtype H5N1 exposure in swine

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzymelinked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses

Instituting an Educational Module to Improve Knowledge of Local Anesthetic Systemic Toxicity Among Anesthesia Providers

Local anesthetic use in the perioperative period is increasing in the United States as healthcare providers seek alternatives to opioid-based pain management. While increased use of local anesthetics has many benefits, their use also increases the risk of Local Anesthetic Systemic Toxicity (LAST), which is a serious and potentially life-threatening complication. A search of the literature indicates there is a considerable gap in knowledge of LAST among healthcare providers, including Certified Registered Nurse Anesthetists (CRNAs) and anesthesia residents. This knowledge gap is combined with a lack of standardized approaches to identifying and treating this complication. Identifying and treating LAST is also complicated by the varying presentations of LAST, including presenting as a stroke mimic. To mitigate this knowledge deficit, we identified a surgical center that has recently increased its use of local anesthetics without increasing education on LAST recognition and management. We developed an online module to educate CRNAs and anesthesia residents about LAST and evaluated changes in their knowledge of LAST with a pre- and post-survey. Statistical analysis of pre- and post-surveys indicated a significant increase in knowledge of LAST

Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling

Alternative promoters drive human cytomegalovirus reactivation from latency

Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner

Radiative Flux and Forcing Parameterization Error in Aerosol-Free Clear Skies

This article reports on the accuracy in aerosol- and cloud-free conditions of the radiation parameterizations used in climate models. Accuracy is assessed relative to observationally validated reference models for fluxes under present-day conditions and forcing (flux changes) from quadrupled concentrations of carbon dioxide. Agreement among reference models is typically within 1 W/m2, while parameterized calculations are roughly half as accurate in the longwave and even less accurate, and more variable, in the shortwave. Absorption of shortwave radiation is underestimated by most parameterizations in the present day and has relatively large errors in forcing. Error in present-day conditions is essentially unrelated to error in forcing calculations. Recent revisions to parameterizations have reduced error in most cases. A dependence on atmospheric conditions, including integrated water vapor, means that global estimates of parameterization error relevant for the radiative forcing of climate change will require much more ambitious calculations

Turning I into me: Imagining your future self.

A widely endorsed belief is that perceivers imagine their present selves using a different representational format than imagining their future selves (i.e., near future=first-person; distant future=third-person). But is this really the case? Responding to the paucity of work on this topic, here we considered how temporal distance influences the extent to which individuals direct their attention outward or inward during a brief imaginary episode. Using a non-verbal measure of visual perspective taking (i.e., letter-drawing task) our results confirmed the hypothesized relation between temporal distance and conceptions of the self. Whereas simulations of an event in the near future were dominated by a first-person representation of the self, this switched to a third-person depiction when the event was located in the distant future. Critically, this switch in vantage point was restricted to self-related simulations. The theoretical and practical implications of these findings are considered

Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases