White-tailed deer (Odocoileus virginianus) may serve as a wildlife reservoir for nearly extinct SARS-CoV-2 variants of concern.

The spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (WTD) and its ability to transmit from deer to deer raised concerns about the role of WTD in the epidemiology and ecology of the virus. Here, we present a comprehensive cross-sectional study assessing the prevalence, genetic diversity, and evolution of SARS-CoV-2 in WTD in the State of New York (NY). A total of 5,462 retropharyngeal lymph node samples collected from free-ranging hunter-harvested WTD during the hunting seasons of 2020 (Season 1, September to December 2020, n = 2,700) and 2021 (Season 2, September to December 2021, n = 2,762) were tested by SARS-CoV-2 real-time RT-PCR (rRT-PCR). SARS-CoV-2 RNA was detected in 17 samples (0.6%) from Season 1 and in 583 samples (21.1%) from Season 2. Hotspots of infection were identified in multiple confined geographic areas of NY. Sequence analysis of SARS-CoV-2 genomes from 164 samples demonstrated the presence of multiple SARS-CoV-2 lineages and the cocirculation of three major variants of concern (VOCs) (Alpha, Gamma, and Delta) in WTD. Our analysis suggests the occurrence of multiple spillover events (human to deer) of the Alpha and Delta lineages with subsequent deer-to-deer transmission and adaptation of the viruses. Detection of Alpha and Gamma variants in WTD long after their broad circulation in humans in NY suggests that WTD may serve as a wildlife reservoir for VOCs no longer circulating in humans. Thus, implementation of continuous surveillance programs to monitor SARS-CoV-2 dynamics in WTD is warranted, and measures to minimize virus transmission between humans and animals are urgently needed.

Hijacking of RIG-I signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type I interferon responses.

UNLABELLED: Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses. To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-ß) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses. IMPORTANCE: The mechanism by which the newly described SFTSV inhibits host antiviral responses has not yet been fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with the inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.

Identification of an Immunodominant B-Cell Epitope in African Swine Fever Virus p30 Protein and Evidence of p30 Antibody-Mediated Antibody Dependent Cellular Cytotoxicity.

African Swine Fever Virus (ASFV) is a large dsDNA virus that encodes at least 150 proteins. The complexity of ASFV and lack of knowledge of effector immune functions and protective antigens have hindered the development of safe and effective ASF vaccines. In this study, we constructed four Orf virus recombinant vectors expressing individual ASFV genes B602L, -CP204L, E184L, and -I73R (ORFVΔ121-ASFV-B602L, -CP204L, -E184L, and -I73R). All recombinant viruses expressed the heterologous ASFV proteins in vitro. We then evaluated the immunogenicity of the recombinants by immunizing four-week-old piglets. In two independent animal studies, we observed high antibody titers against ASFV p30, encoded by CP204L gene. Using Pepscan ELISA, we identified a linear B-cell epitope of 12 amino acids in length (Peptide 15) located in an exposed loop region of p30 as an immunodominant ASFV epitope. Additionally, antibodies elicited against ASFV p30 presented antibody-dependent cellular cytotoxicity (ADCC) activity. These results underscore the role of p30 on antibody responses elicited against ASFV and highlight an important functional epitope that contributes to p30-specific antibody responses.

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, an mAb-based blocking enzyme-linked immunosorbent assay (bELISA) was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 4.89%, and 3.16%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with coronavirus disease 2019 (COVID-19)-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCE Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid (N) protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, a mAb-based bELISA was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition (PI) cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 6.95%, and 5.15%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with COVID-19-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCE: Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

Detection and characterization of a distinct bornavirus lineage from healthy Canada geese (Branta canadensis).

Avian bornaviruses (ABV), identified in 2008, infect captive parrots and macaws worldwide. The natural reservoirs of these viruses are unknown. Reverse transcription-PCR (RT-PCR) was used to screen oropharyngeal/cloacal swab and brain samples from wild Canada geese (Branta canadensis) for ABV. Approximately 2.9% of swab samples were positive for bornavirus sequences. Fifty-two percent of brain samples from 2 urban flocks also tested positive, and brain isolates were cultured in duck embryo fibroblasts. Phylogenetic analyses placed goose isolates in an independent cluster, and more notably, important regulatory sequences present in Borna disease virus but lacking in psittacine ABVs were present in goose isolates.

FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1.

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK.

Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum.

Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1x coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40-70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP-dROX, we assembled a physical map for P.chrysogenum, with 2-3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.

Cloning, characterization, and evolution of the NBS-LRR-encoding resistance gene analogue family in polyploid cotton (Gossypium hirsutum L.).

The nucleotide-binding site-leucine-rich repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. We cloned the NBS-LRR-encoding genes from polyploid cotton by a polymerase chain reaction-based approach. A sample of 150 clones was selected from the NBS-LRR gene sequence library and was sequenced, and 61 resistance gene analogs (RGA) were identified. Sequence analysis revealed that RGA are abundant and highly diverged in the cotton genome and could be categorized into 10 distinct subfamilies based on the similarities of their nucleotide sequences. The numbers of members vary many fold among different subfamilies, and gene index analysis showed that each of the subfamilies is at a different stage of RGA family evolution. Genetic mapping of a selection of RGA indicates that the RGA reside on a limited number of the cotton chromosomes, with those from a single subfamily tending to cluster and two of the RGA loci being colocalized with the cotton bacterial blight resistance genes. The distribution of RGA between the two subgenomes A and D of cotton is uneven, with RGA being more abundant in the A subgenome than in the D subgenome. The data provide new insights into the organization and evolution of the NBS-LRR-encoding RGA family in polyploid plants.

Widespread avian bornavirus infection in mute swans in the Northeast United States.

Avian bornavirus (ABV) matrix (M) genes were detected by RT-PCR on brain tissue obtained from 192 mute swans harvested from several Northeastern states. A RT-PCR product was detected in 45 samples. Sequencing of the PCR products confirmed the presence of ABV belonging to the 'goose' genotype. The prevalence of positive samples ranged from 28% in Michigan to 0% in northern New York State. Two Rhode Island isolates were cultured. Their M, N, and X-P gene sequences closely matched recently published sequences from Canada geese.

EIAV S2 enhances pro-inflammatory cytokine and chemokine response in infected macrophages.

Equine infectious anemia virus (EIAV) infection is distinctive in that it causes a rapid onset of clinical disease relative to other retroviruses. In order to understand the interaction dynamics between EIAV and the host immune response, we explored the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine response in macrophages. EIAV infection markedly altered the expression pattern of a variety of pro-inflammatory cytokines and chemokines monitored in the study. Comparative studies in the cytokine response between EIAV(17) and EIAV(17DeltaS2) infection revealed that S2 enhances the expression of IL-1alpha, IL-1beta, IL-8, MCP-2, MIP-1beta and IP-10. Moreover, S2 specifically induced the expression of the newly discovered cytokine, IL-34. Taken together, these results may help explain the effect of cytokine and chemokine dysregulation in EIAV pathogenesis and suggest a role of S2 in optimizing the host cell environment to promote viral dissemination and replication.

Identification of cellular proteins interacting with equine infectious anemia virus S2 protein.

The macrophage-tropic lentivirus, equine infectious anemia virus (EIAV), encodes the small auxiliary protein S2 from a short open reading frame that overlaps the amino terminus of env EIAV S2 is dispensable for virus replication in cultured cells but is required for disease production. S2 is approximately 7 kDa and has no overall amino acid sequence homology to other cellular or viral proteins. Therefore it is likely that S2 plays a role as an adaptor protein. To further investigate S2 function we performed a yeast-2-hybrid screen to identify cellular proteins that interact with EIAV S2. The screen identified two human cellular proteins, amplified in osteosarcoma (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) that interact with S2. The equine homologues of these proteins were cloned and their interactions with S2 confirmed using co-immunoprecipitation assays. We identified two OS-9 isoforms that interact with S2 and a third splice variant that does not, indicating a region of OS-9 apparently required for the S2 interaction. The roles of these cellular proteins during EIAV infection have not been determined.

Genome physical mapping with large-insert bacterial clones by fingerprint analysis: methodologies, source clone genome coverage, and contig map quality.

Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.