Seroprevalence of SARS-CoV-2 antibodies among rural healthcare workers.

The objective of this longitudinal cohort study was to determine the seroprevalence of antibodies to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in healthcare workers employed at healthcare settings in three rural counties in eastern South Dakota and western Minnesota from May 13, 2020, through December 22, 2020. Three blood draws were performed at five clinical sites and tested for the presence of antibodies against the SARS-CoV-2. Serum samples were tested for the presence of antibodies using a fluorescent microsphere immunoassay (FMIA), neutralization of SARS-CoV-2 spike-pseudotyped particles (SARS-CoV-2pp) assay, and serum virus neutralization (SVN) assay. The seroprevalence was determined to be 1/336 (0.29%) for samples collected from 5/13/20 to 7/13/20, 5/260 (1.92%) for samples collected from 8/13/20 to 9/25/20, and 35/235 (14.89%) for samples collected from 10/16/20 to 12/22/20. Eight of the 35 (22.8%) seropositive individuals identified in the final draw did not report a previous diagnosis with COVID-19. There was a high correlation (>90%) between the FMIA and virus neutralization assays. Each clinical site's seroprevalence was higher than the cumulative incidence for the general public in the respective county as reported by state public health agencies. As of December 2020, there was a high percentage (85%) of seronegative individuals in the study population.

Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses.

BACKGROUND AND OBJECTIVES: Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. METHODS AND RESULTS: Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. CONCLUSION: This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.

Targeted mutations in a highly conserved motif of the nsp1ß protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus.

Non-structural protein 1ß (nsp1ß) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a papain-like cysteine protease (PLPß) domain and has been identified as the main viral protein antagonizing the host innate immune response. In this study, nsp1ß was determined to suppress the expression of reporter genes as well as to suppress 'self-expression' in transfected cells, and this activity appeared to be associated with its interferon (IFN) antagonist function. To knock down the effect of nsp1ß on IFN activity, a panel of site-specific mutations in nsp1ß was analysed. Double mutations K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV) targeting a highly conserved motif of nsp1ß, GKYLQRRLQ (in bold), impaired the ability of nsp1ß to suppress IFN-ß and reporter gene expression, as well as to suppress 'self-expression' in vitro. Subsequently, viable recombinant viruses vSD01-08-K130A/R134A and vSD95-21-K124A/R128A, containing double mutations in the GKYLQRRLQ motif were generated using reverse genetics. In comparison with WT viruses, these nsp1ß mutants showed impaired growth ability in infected cells, but the PLPß cleavage function was not directly affected. The expression of selected innate immune genes was determined in vSD95-21-K124A/R128A mutant-infected cells. The results consistently showed that gene expression levels of IFN-α, IFN-ß and IFN-stimulated gene 15 were upregulated in cells that were infected with the vSD95-21-K124A/R128A compared with that of WT virus. These data suggest that PRRSV nsp1ß may selectively suppress cellular gene expression, including expression of genes involved in the host innate immune function. Modifying the key residues in the highly conserved GKYLQRRLQ motif could attenuate virus growth and improve the cellular innate immune responses.

Interleukin-1ß expression by a recombinant porcine reproductive and respiratory syndrome virus.

The cytokine interleukin-1 beta (IL-1ß) is a potent inflammatory mediator in response to infection, and can be used as an immunological adjuvant. In this study, we constructed a recombinant porcine reproductive and respiratory syndrome virus (vP129/swIL1ß) expressing swine IL-1ß from the separate subgenomic mRNA inserted between the ORF1b and ORF2 genome region. MARC-145 cells infected with vP129/swIL1ß secreted 1947 pg of IL-1ß per 2 × 10(5)cells at 36 h post-infection. In vitro growth kinetics analysis in MARC-145 cells showed that the vP129/swIL1ß virus had a similar replication rate as that of parental virus. We further performed in vivo characterization of the vP129/swIL1ß virus in a nursery pig disease model. The vP129/swIL1ß infected pigs did not show visible clinical signs, while respiratory distress and lethargy were evident in pigs infected with the parental virus. The expression of various cytokines from peripheral blood mononuclear cells measured by fluorescent microsphere immunoassay showed that IL-1ß, IL-4 and IFN-γ expression levels were up-regulated in pigs infected with vP129/swIL1ß at 7 and 14 days post-infection. However, no detectable level of IL-1ß was found in serum samples from pigs infected with either vP129/swIL1ß or parental virus. In summary, this study demonstrated a recombinant PRRSV as a useful tool to study the role of different cytokines in disease progression and immune responses, which represents a new strategy for future therapeutic application and vaccine development.

The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions.

Porcine reproductive and respiratory syndrome (PRRS) virus nonstructural protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-kappaB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-kappaB activation by interfering with the polyubiquitination process of IkappaBalpha, which subsequently prevents IkappaBalpha degradation. To determine whether the nsp2 protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-kappaB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-kappaB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-kappaB activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.

Development of genetic markers in the non-structural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem in the pork industry worldwide. The limitations of current PRRSV vaccines require the development of a new generation of vaccines. One of the key steps in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of type 1 PRRSV, this study constructed a recombinant green fluorescent protein (GFP)-tagged PRRSV containing a deletion of an immunogenic epitope, ES4, in the nsp2 region. In a nursery pig disease model, the recombinant virus was attenuated with a lower level of viraemia in comparison with that of the parental virus. To complement the marker identification, GFP and ES4 epitope-based ELISAs were developed. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, but generated a high-level antibody response to GFP by 14 days post-infection. These results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation between marker virus-infected animals and those infected with the wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRS.

The 2b protein as a minor structural component of PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) ORF2 contains an internal ORF that codes for a small non-glycosylated protein known as 2b. Previous work had identified the presence of a 10kDa 2b protein in virus-infected cells and the induction of an anti-2b response in PRRSV-infected pigs, as well as a possible association of 2b with the virion (, Virology 287:183-191). In this study, we utilized two experimental approaches, including the use of a 2b peptide-specific monoclonal antibody, to demonstrate that the PRRSV 2b protein is an integral component of the PRRSV virion. This study suggests that 2b in PRRSV is similar to the E protein in EAV and forms a minor structural component of the virion.

Proactive monitoring and adaptive management of social carrying capacity in Arches National Park: an application of computer simulation modeling.

Public visits to parks and protected areas continue to increase and may threaten the integrity of natural and cultural resources and the quality of the visitor experience. Scientists and managers have adopted the concept of carrying capacity to address the impacts of visitor use. In the context of outdoor recreation, the social component of carrying capacity refers to the level of visitor use that can be accommodated in parks and protected areas without diminishing the quality of the visitor experience to an unacceptable degree. This study expands and illustrates the use of computer simulation modeling as a tool for proactive monitoring and adaptive management of social carrying capacity at Arches National Park. A travel simulation model of daily visitor use throughout the Park's road and trail network and at selected attraction sites was developed, and simulations were conducted to estimate a daily social carrying capacity for Delicate Arch, an attraction site in Arches National Park, and for the Park as a whole. Further, a series of simulations were conducted to estimate the effect of a mandatory shuttle bus system on daily social carrying capacity of Delicate Arch to illustrate how computer simulation modeling can be used as a tool to facilitate adaptive management of social carrying capacity.

Carrying capacity as "Informed Judgment": the values of science and the science of values.

Contemporary park and wilderness carrying capacity frameworks rely on formulation of standards of quality, which are defined as minimum acceptable resource and social conditions. Formulation of standards of quality involves elements of both science and values, and both of these elements must be integrated into informed judgments on the part of park and wilderness managers. That is, managers must ultimately make value-based judgments about the maximum acceptable level of visitor-caused impacts to the resource base and the quality of the visitor experience. However, such judgments should be as informed as possible by scientific data on the relationships between visitor use and resulting impacts and the degree to which park and wilderness visitors and other interest groups judge such impacts to be acceptable. Such information represents the "values of science" to managing carrying capacity in parks and wilderness. A growing body of literature has begun to address the corresponding "science of values," and how this type of information might be integrated in park and wilderness management. Visitor-based research has employed normative theory and techniques to explore the acceptability of a range of resource and social impacts related to visitor use, and findings from these studies are being integrated into a body of knowledge and applied in management decision-making. Conceptual and methodological extensions of the normative approach are currently being explored in a variety of park and wilderness contexts, and new theoretical and empirical approaches are being adapted to address trade-offs inherent in carrying capacity. In these ways, the science of values is progressing to meet the opportunities and challenges of the values of science to park and wilderness management. The concept of carrying capacity, along with the theoretical and methodological approaches described in this paper, can be extended to a large number of natural resource and environmental issues.