Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories.

Introduction: Pathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation. Methods: SARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications. Results: Heat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing. Discussion: By ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed.

Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants.

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.

The Convergence of High-Consequence Livestock and Human Pathogen Research and Development: A Paradox of Zoonotic Disease.

The World Health Organization (WHO) estimates that zoonotic diseases transmitted from animals to humans account for 75 percent of new and emerging infectious diseases. Globally, high-consequence pathogens that impact livestock and have the potential for human transmission create research paradoxes and operational challenges for the high-containment laboratories that conduct work with them. These specialized facilities are required for conducting all phases of research on high-consequence pathogens (basic, applied, and translational) with an emphasis on both the generation of fundamental knowledge and product development. To achieve this research mission, a highly-trained workforce is required and flexible operational methods are needed. In addition, working with certain pathogens requires compliance with regulations such as the Centers for Disease Control (CDC) and the U.S. Department of Agriculture (USDA) Select Agent regulations, which adds to the operational burden. The vast experience from the existing studies at Plum Island Animal Disease Center, other U.S. laboratories, and those in Europe and Australia with biosafety level 4 (BSL-4) facilities designed for large animals, clearly demonstrates the valuable contribution this capability brings to the efforts to detect, prepare, prevent and respond to livestock and potential zoonotic threats. To raise awareness of these challenges, which include biosafety and biosecurity issues, we held a workshop at the 2018 American Society for Microbiology (ASM) Biothreats conference to further discuss the topic with invited experts and audience participants. The workshop covered the subjects of research funding and metrics, economic sustainment of drug and vaccine development pipelines, workforce turnover, and the challenges of maintaining operational readiness of high containment laboratories.

High-impact animal health research conducted at the USDA's National Animal Disease Center.

Commissioned by President Dwight Eisenhower in 1958 and opened with a dedication ceremony in December 1961, the USDA, Agricultural Research Service (ARS), National Animal Disease Center (NADC) celebrated its 50-year anniversary in November 2011. Over these 50 years, the NADC established itself among the world's premier animal health research centers. Its historic mission has been to conduct basic and applied research on selected endemic diseases of economic importance to the U.S. livestock and poultry industries. Research from NADC has impacted control or management efforts on nearly every major animal disease in the United States since 1961. For example, diagnostic tests and vaccines developed by NADC scientists to detect and prevent hog cholera were integral in the ultimate eradication of this costly swine disease from the U.S. Most major veterinary vaccines for critical diseases such as brucellosis and leptospirosis in cattle, porcine respiratory and reproductive syndrome (PRRS), porcine parvovirus and influenza in swine had their research origins or were developed and tested at the NADC. Additional discoveries made by NADC scientists have also resulted in the development of a nutritional approach and feed additives to prevent milk fever in transition dairy cattle. More recently, NADC's archive of historic swine influenza viruses combined with an established critical mass of influenza research expertise enabled NADC researchers to lead an effective national research response to the pandemic associated with the novel 2009 H1N1 influenza virus. This review commemorates some of the key animal health contributions in NADC's first 50 years, recaps the newly completed modernization of the center into new facilities, and offers highlights of the ongoing research that will define NADC's mission going forward.

Comparative transcriptome analysis of in vivo- and in vitro-produced porcine blastocysts by small amplified RNA-serial analysis of gene expression (SAR-SAGE).

Production of embryos in vitro has enormous potential for research and commercial applications. Unfortunately, in vitro production of porcine embryos is extremely inefficient. Despite the characterization of distinct phenotypes, little is known about the molecular mechanisms and altered physiological processes of in vitro-produced embryos. The objective of this study was to compare global gene expression patterns from in vivo- (IVO) and in vitro-produced (IVP) porcine embryos using small amplified RNA-serial analysis of gene expression (SAR-SAGE). Whole-cell RNA from pools of Day 6 IVO and IVP blastocysts was used to construct SAR-SAGE libraries. Sequence analysis of the IVO and IVP libraries yielded 98,771 and 98,408 tags, respectively. A total of 20,029 and 23,453 putative transcripts were detected in the IVO and IVP libraries, respectively. Statistical analyses of SAGE tag frequencies between the IVO and IVP libraries indicated that 938 and 193 tags were differentially expressed at a P < 0.05 and P < 0.001 level of significance, respectively, suggesting significant deviations in transcriptome profiles from IVO and IVP embryos. Categorization of differentially expressed transcripts into functional groupings indicated a significant deviation in gene expression from IVP blastocysts compared with IVO blastocysts for a number of biological processes including cellular metabolism, organization, and response to stress. Real-time PCR confirmed differential expression for several transcripts from independent IVO and IVP blastocysts. These results demonstrate compromised gene expression in IVP blastocysts compared with IVO blastocysts for a number of biological processes, particularly processes involved in mitochondrial function; thereby providing potential target pathways for improvement of IVP methods.

Transcriptome profiling of the tubular porcine conceptus identifies the differential regulation of growth and developmentally associated genes.

Gastrulation and trophectoderm elongation of the porcine conceptus coincide with peak conceptus estrogen secretion from gestational day 11 to day 12. The current study aim was to identify genes required for elongation by defining the transcriptome profile of this dynamic tubular stage. The gastrulation and proliferative status of ovoid, tubular, and filamentous conceptuses were also examined. Polarization of the embryonic disc and growth throughout the conceptus were evident. An unamplified and two distinct amplified serial analysis of gene expression (SAGE) libraries were generated from tubular conceptus mRNA. Comparing the three libraries at 12,000 tags/library indicated small-amplified RNA-SAGE was a reliable amplification procedure. The unamplified library was increased to 42,415 tags and statistical analyses of tag frequencies with previously generated ovoid and filamentous libraries revealed the differential expression (P < 0.05) of 483 and 364 tags between ovoid:tubular or tubular:filamentous libraries, respectively. Annotated transcripts known to be involved in development and also potentially regulated by estrogen (cytokeratins 8 and 18, stratifin, midkine, and glycolytic enzymes) were further analyzed by real-time PCR. The majority of glycolytic enzyme transcripts were constitutively expressed or downregulated at the filamentous stage. Likewise, cytokeratin mRNAs were less abundant in filamentous conceptuses, whereas stratifin and midkine were more abundant in tubular conceptuses. Analysis of protein revealed distinct expression patterns for cytokeratin 18, stratifin, and midkine. The function(s) of these factors and potential modulation by estrogen clearly needs to be elucidated to understand their physiological role in normal conceptus development.

Expression analysis of the steroidogenic acute regulatory protein (STAR) gene in developing porcine conceptuses.

Steroidogenesis in porcine non-conceptus tissue is regulated by the STAR-dependent transport of cholesterol from the outer to inner mitochondrial membrane. Previous serial analysis of gene expression (SAGE) identified a STAR mRNA transcript in the porcine peri-implantation conceptus during trophectoderm elongation and increased conceptus estrogen synthesis between gestational day 11 and 12. To assess a potential role for STAR in the modulation of conceptus steroidogenesis via cholesterol transport, the conceptus STAR transcript was PCR cloned and temporal expression of mRNA and protein were examined. Northern analysis of day 12 corpora lutea and pig conceptus RNA detected multiple STAR transcripts in both tissues and identified the cloned transcript as the longest variant. The transcript had a 99% similarity to a truncated ovarian STAR transcript. The conceptus STAR transcript was temporally regulated during elongation but trace expression was present in day 6 blastocysts and day 25 conceptuses. Differential regulation of STAR mRNA was concomitant with the presence of the stimulatory transcription factor steroidogenic factor 1, and absence of the inhibitory transcription factor dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1, transcripts. In contrast to peak STAR mRNA expression at the filamentous stage, Western blot analyses revealed STAR protein levels were highest in tubular conceptuses. These data confirm the presence of STAR mRNA and protein during porcine conceptus elongation and suggest regulation of STAR at two levels, transcriptionally, in part, through differential regulation of transcription factors, and post-transcriptionally, evidenced by the disparity of protein to RNA in filamentous conceptuses.

Serial analysis of gene expression during elongation of the peri-implantation porcine trophectoderm (conceptus).

Conceptus loss during the preimplantation and early postimplantation period hinders the efficiency of swine reproduction. Significant conceptus loss occurs during trophectoderm elongation between gestational day 11 (D11) and day 12 (D12). Elongation of the porcine conceptus is a key stage of development during which maternal recognition of pregnancy, initial placental development, and preparation for implantation occurs. The objective of this study was to establish comparative transcriptome profiles of D11 ovoid and D12 filamentous conceptuses and thereby identify temporally regulated genes essential for developmental progression during conceptus elongation. Serial analysis of gene expression (SAGE) libraries were constructed from in vivo derived ovoid and filamentous swine conceptuses to yield a total of 42,389 tags (ovoid) and 42,391 tags (filamentous) representing 14,464 and 13,098 putative unique transcripts, respectively. Statistical analysis of tag frequencies revealed the differential expression of 431 tags between libraries (P < 0.05). Nucleotide sequence alignment searches on public databases provided SAGE tag annotation and gene ontology assignments. Comparisons between the SAGE profiles of ovoid and filamentous conceptuses revealed increased expression of key genes in the steroidogenesis [cytochrome P-450(scc) (CYP11A1), aromatase (CYP19A), and steroidogenic acute regulatory protein (STAR)] and oxidative stress response pathways [microsomal glutathione S-transferase 1 (MGST1) and copper-zinc superoxide dismutase (SOD1)]. Differential expression of these genes in the steroidogenic and oxidative stress response pathways was confirmed by real-time PCR. These results validate the utility of SAGE in the pig and establish an initial model linking gene expression profiles at the pathway level with phenotypic progression from ovoid to filamentous stages of conceptus development.

Serial analysis of gene expression (SAGE) during porcine embryo development.

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

Serial analysis of gene expression in turkey sperm storage tubules in the presence and absence of resident sperm.

Turkey sperm lose viability within 8-18 h when stored as liquid semen using current methods and extenders. In contrast, turkey hens maintain viable, fertile sperm in their sperm storage tubules (SST) for 45 or more days following a single insemination. Our long-term objectives are to identify and characterize differentially expressed genes that may underlie this prolonged sperm storage and then use this information to develop improved methods for storing liquid turkey semen. We employed serial analysis of gene expression (SAGE) to compare gene expression patterns in turkey SST recovered from hens after artificial insemination (AI) with extended semen (sperm AI) or extender alone (control AI). We constructed two separate SAGE libraries with SST RNA obtained from sperm and control AI hens. We used these libraries to generate 95,325 ten-base pair SAGE tags. These 95,325 tags represented 27,430 unique genes. The sperm and control AI libraries contained 47,663 and 47,662 tags representing 18,030 and 19,101 putative unique transcripts, respectively. Approximately 1% of these putative unique genes were differentially expressed (P<0.05) between treatments. Tentative annotations were ascribed to the SAGE tag nucleotide sequences by comparing them against publicly available SAGE tag and cDNA sequence databases. Based on its SAGE tag nucleotide sequence, we cloned a partial turkey avidin cDNA and confirmed its up-regulation in the sperm AI SST. The bioinformatics and experimental procedures employed to clone turkey avidin and confirm its differential expression represent a useful paradigm for analyzing SAGE tag data from relatively uncharacterized model systems.

Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes, zygotes, and pre-implantation stage embryos.

Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature oocytes and decline after fertilization, suggest that GSH synthesis may be associated with cell cycle events. To explore this possibility, we measured the concentrations of GSH in Golden Hamster oocytes and zygotes at specific stages of oocyte maturation and at intervals during the first complete embryonic cell cycle. Between 2 and 4 hr after the hormonal induction of oocyte maturation, GSH concentrations increased significantly (approximately doubling) in both oocytes and their associated cumulus cells. This increase was concurrent with germinal vesicle breakdown and the condensation of metaphase I chromosomes in the oocyte. GSH remained high in ovulated, metaphase II (MII) oocytes, but then declined significantly, by about 50%, shortly after fertilization, as the zygote progressed back into interphase (the pronucleus stage). GSH concentrations then plummeted by the two-cell embryo stage and remained at only 10% of those in MII oocytes throughout pre-implantation development. These results demonstrate that oocyte GSH concentrations fluctuate with the cell cycle, being highest during meiotic metaphase, the critical period for spindle growth and development and for sperm chromatin remodeling. These observations raise the possibility that GSH synthesis in maturing oocytes is regulated by gonadotropins, and suggest that GSH is more important during fertilization than during pre-implantation embryo development.