Review of Restricted Experiment Requests, Division of Select Agents and Toxins, US Centers for Disease Control and Prevention, 2014-2021.

The US Centers for Disease Control and Prevention Division of Select Agents and Toxins (DSAT) regulates laboratories that possess, use, or transfer select agents and toxins within United States as part of the Federal Select Agent Program. DSAT also mitigates biosafety risks through the review of "restricted experiments," which under the select agent regulations are experiments that pose heightened biosafety risks. In a previous study, we evaluated restricted experimental requests submitted to DSAT for review between 2006 and 2013. The purpose of this study is to provide an updated analysis of requests to conduct potential restricted experiments submitted to DSAT between 2014 and 2021. This article describes the trends and characteristics of the data associated with restricted experimental requests involving select agents and toxins that have an impact on public health and safety (US Department of Health and Human Services agents only) or both public health and safety and animal health or products (overlap agents). From January 2014 to December 2021, DSAT received 113 requests to conduct potential restricted experiments; however, 82% (n=93) of those requests were determined not to meet the regulatory definition of a restricted experiment. Of the 20 requests that met the definition of a restricted experiment, 8 were denied because the experiments had the potential to compromise disease control in humans. DSAT continues to encourage entities to practice due diligence and request a review of research that could potentially meet the regulatory definition of a restricted experiment out of an abundance of caution to protect public health and safety and prevent any potential compliance action.

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2.

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hypertrophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocardiography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Resolution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell-cycle defects and mitochondrial dysfunction. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteasomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteasomal system is activated; a mechanism that has been implicated in cardiomyopathies previously. In parallel, a lack of functional alpha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell-cycle defects, the likely cause of the death of the embryos. The defects also have wide-ranging morphological consequences.

Cytokines and chemokines: The vital role they play in herpes simplex virus mucosal immunology.

Herpes simplex viruses (HSV) types 1 and 2 are ubiquitous infections in humans. They cause orofacial and genital herpes with occasional severe complications. HSV2 also predisposes individuals to infection with HIV. There is currently no vaccine or immunotherapy for these diseases. Understanding the immunopathogenesis of HSV infections is essential to progress towards these goals. Both HSV viruses result in initial infections in two major sites - in the skin or mucosa, either after initial infection or recurrence, and in the dorsal root or trigeminal ganglia where the viruses establish latency. HSV1 can also cause recurrent infection in the eye. At all of these sites immune cells respond to control infection. T cells and resident dendritic cells (DCs) in the skin/mucosa and around reactivating neurones in the ganglia, as well as keratinocytes in the skin and mucosa, are major sources of cytokines and chemokines. Cytokines such as the Type I and II interferons synergise in their local antiviral effects. Chemokines such as CCL2, 3 and 4 are found in lesion vesicle fluid, but their exact role in determining the interactions between epidermal and dermal DCs and with resident memory and infiltrating CD4 and CD8 T cells in the skin/mucosa is unclear. Even less is known about these mechanisms in the ganglia. Here we review the data on known sources and actions of these cytokines and chemokines at cellular and tissue level and indicate their potential for preventative and therapeutic interventions.

Characterization of Lymphatic Vasculature Using Whole-Mount Immunostaining of Mouse Embryonic Dorsal Skin.

Understanding the development of the lymphatic vasculature is essential to the understanding of how these vessels function in health and disease. High-resolution imaging of histological techniques such as immunostaining of sectioned tissue provides a snapshot into lymphatic vessel morphogenesis, patterning, and organization. Whole-mount staining of embryonic dermal vasculature allows for a deeper analysis and characterization of the developing lymphatic vascular network.

Maternal choline supplementation in a rat model of periconceptional alcohol exposure: Impacts on the fetus and placenta.

BACKGROUND: Maternal choline supplementation in rats can ameliorate specific neurological and behavioral abnormalities caused by alcohol exposure during pregnancy. We tested whether choline supplementation ameliorates fetal growth restriction and molecular changes in the placenta associated with periconceptional ethanol exposure (PCE) in the rat. METHODS: Sprague Dawley dams were given either 12.5% ethanol (PCE) or 0% ethanol (Con) in a liquid diet from 4 days prior to 4 days after conception. At day 5 of pregnancy, dams were either placed on a standard chow (1.6 g choline/kg chow) or an intermediate chow (2.6 g choline/kg chow). On day 10 of pregnancy, a subset of the intermediate dams were placed on a chow further supplemented with choline (7.2 g choline/kg chow), resulting in 6 groups. Fetuses and placentas were collected on day 20 of pregnancy for analysis. RESULTS: Choline supplementation resulted in increased fetal weight at late gestation, ameliorating the deficits due to PCE. This was most pronounced in litters on a standard chow during pregnancy. Choline also increased fetal liver weight and decreased fetal brain:liver ratio, independent of alcohol exposure. Placental weight was reduced as choline levels in the chow increased, particularly in female placentas. This resulted in a greater ratio of fetal:placental weight, suggesting increased placental efficiency. Global DNA methylation in the placenta was altered in a sex-specific manner by both PCE and choline. However, the increased glycogen deposition in female placentas, previously reported in this PCE model, was not prevented by choline supplementation. CONCLUSIONS: Our results suggest that choline has the potential to ameliorate fetal growth restriction associated with PCE and improve placental efficiency following prenatal alcohol exposure. Our study highlights the importance of maternal nutrition in moderating the severity of adverse fetal and placental outcomes that may arise from prenatal alcohol exposure around the time of conception.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.

Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

Analysis of Placental Arteriovenous Formation Reveals New Insights Into Embryos With Congenital Heart Defects.

The placental vasculature provides the developing embryo with a circulation to deliver nutrients and dispose of waste products. However, in the mouse, the vascular components of the chorio-allantoic placenta have been largely unexplored due to a lack of well-validated molecular markers. This is required to study how these blood vessels form in development and how they are impacted by embryonic or maternal defects. Here, we employed marker analysis to characterize the arterial/arteriole and venous/venule endothelial cells (ECs) during normal mouse placental development. We reveal that placental ECs are potentially unique compared with their embryonic counterparts. We assessed embryonic markers of arterial ECs, venous ECs, and their capillary counterparts-arteriole and venule ECs. Major findings were that the arterial tree exclusively expressed Dll4, and venous vascular tree could be distinguished from the arterial tree by Endomucin (EMCN) expression levels. The relationship between the placenta and developing heart is particularly interesting. These two organs form at the same stages of embryogenesis and are well known to affect each other's growth trajectories. However, although there are many mouse models of heart defects, these are not routinely assessed for placental defects. Using these new placental vascular markers, we reveal that mouse embryos from one model of heart defects, caused by maternal iron deficiency, also have defects in the formation of the placental arterial, but not the venous, vascular tree. Defects to the embryonic cardiovascular system can therefore have a significant impact on blood flow delivery and expansion of the placental arterial tree.

Environmental Risk Factors for Congenital Heart Disease.

Congenital heart disease (CHD) has many forms and a wide range of causes. Clinically, it is important to understand the causes. This allows estimation of recurrence rate, guides treatment options, and may also be used to formulate public health advice to reduce the population prevalence of CHD. The recent advent of sophisticated genetic and genomic methods has led to the identification of more than 100 genes associated with CHD. However, despite these great strides, to date only one-third of CHD cases have been shown to have a simple genetic cause. This is because CHD can also be caused by oligogenic factors, environmental factors, and/or gene-environment interaction. Although solid evidence for environmental causes of CHD have been available for almost 80 years, it is only very recently that the molecular mechanisms for these risk factors have begun to be investigated. In this review, we describe the most important environmental CHD risk factors, and what is known about how they cause CHD.

Vaccines for Herpes Simplex: Recent Progress Driven by Viral and Adjuvant Immunology.

Herpes simplex viruses (HSV) types 1 and 2 are ubiquitous. They both cause genital herpes, occasionally severe disease in the immunocompromised, and facilitate much HIV acquisition globally. Despite more than 60 years of research, there is no licensed prophylactic HSV vaccine and some doubt as to whether this can be achieved. Nevertheless, a previous HSV vaccine candidate did have partial success in preventing genital herpes and HSV acquisition and another immunotherapeutic candidate reduced viral shedding and recurrent lesions, inspiring further research. However, the entry pathway of HSV into the anogenital mucosa and the subsequent cascade of immune responses need further elucidation so that these responses could be mimicked or improved by a vaccine, to prevent viral entry and colonization of the neuronal ganglia. For an effective novel vaccine against genital herpes the choice of antigen and adjuvant may be critical. The incorporation of adjuvants of the vaccine candidates in the past, may account for their partial efficacy. It is likely that they can be improved by understanding the mechanisms of immune responses elicited by different adjuvants and comparing these to natural immune responses. Here we review the history of vaccines for HSV, those in development and compare them to successful vaccines for chicken pox or herpes zoster. We also review what is known of the natural immune control of herpes lesions, via interacting innate immunity and CD4 and CD8 T cells and the lessons they provide for development of new, more effective vaccines.

Periconceptional ethanol exposure induces a sex specific diuresis and increase in AQP2 and AVPR2 in the kidneys of aged rat offspring.

Maternal alcohol consumption can impair renal development and program kidney dysfunction in offspring. Given that most women who drink alcohol cease consumption upon pregnancy recognition, we aimed to investigate the effect of alcohol around the time of conception (PC:EtOH) on offspring renal development and function. Rats received a liquid diet ±12.5% v/v ethanol from 4 days before to 4 days after mating. At postnatal day 30, nephron number was assessed. Urine flow and electrolyte (Na, K, Cl) excretion was measured at 6 and 19 months and blood pressure at 12 months. At 19 months, kidneys were collected for gene and protein analysis and assessment of collecting duct length. At postnatal day 30, PC:EtOH offspring had fewer nephrons. At 6 months, PC:EtOH exposure did not alter urine flow nor affect blood pressure at 12 months. At 19 months, female but not male offspring exposed to PC:EtOH drank more water and had a higher urine flow despite no differences in plasma arginine vasopressin (AVP) concentrations. Aqp2 mRNA and Avpr2 mRNA and protein expression was increased in kidneys from female PC:EtOH offspring but collecting duct lengths were similar. Immunofluorescent staining revealed diffuse cytoplasmic distribution of AQP2 protein in kidneys from PC:EtOH females, compared with controls with apical AQP2 localization. PC:EtOH resulted in a low nephron endowment and in female offspring, associated with age-related diuresis. Changes in expression and cellular localization of AQP2 likely underpin this disturbance in water homeostasis and highlight the need for alcohol to be avoided in early pregnancy.

Periconceptional alcohol exposure causes female-specific perturbations to trophoblast differentiation and placental formation in the rat.

The development of pathologies during pregnancy, including pre-eclampsia, hypertension and fetal growth restriction (FGR), often originates from poor functioning of the placenta. In vivo models of maternal stressors, such as nutrient deficiency, and placental insufficiency often focus on inadequate growth of the fetus and placenta in late gestation. These studies rarely investigate the origins of poor placental formation in early gestation, including those affecting the pre-implantation embryo and/or the uterine environment. The current study characterises the impact on blastocyst, uterine and placental outcomes in a rat model of periconceptional alcohol exposure, in which 12.5% ethanol is administered in a liquid diet from 4 days before until 4 days after conception. We show female-specific effects on trophoblast differentiation, embryo-uterine communication, and formation of the placental vasculature, resulting in markedly reduced placental volume at embryonic day 15. Both sexes exhibited reduced trophectoderm pluripotency and global hypermethylation, suggestive of inappropriate epigenetic reprogramming. Furthermore, evidence of reduced placental nutrient exchange and reduced pre-implantation maternal plasma choline levels offers significant mechanistic insight into the origins of FGR in this model.

Mechanisms of Immune Control of Mucosal HSV Infection: A Guide to Rational Vaccine Design.

Herpes Simplex Virus (HSV) is a highly prevalent sexually transmitted infection that aside from causing cold sores and genital lesions, causes complications in the immunocompromised and has facilitated a large proportion of HIV acquisition globally. Despite decades of research, there is no prophylactic HSV vaccine ready for use in humans, leaving many questioning whether a prophylactic vaccine is an achievable goal. A previous HSV vaccine trial did have partial success in decreasing acquisition of HSV2-promising evidence that vaccines can prevent acquisition. However, there is still an incomplete understanding of the immune response pathways elicited by HSV after initial mucosal infection and how best to replicate these responses with a vaccine, such that acquisition and colonization of the dorsal root ganglia could be prevented. Another factor to consider in the rational design of an HSV vaccine is adjuvant choice. Understanding the immune responses elicited by different adjuvants and whether lasting humoral and cell-mediated responses are induced is important, especially when studies of past trial vaccines found that a sufficiently protective cell-mediated response was lacking. In this review, we discuss what is known of the immune control involved in initial herpes lesions and reactivation, including the importance of CD4 and CD8 T cells, and the interplay between innate and adaptive immunity in response to primary infection, specifically focusing on the viral relay involved. Additionally, a summary of previous and current vaccine trials, including the components used, immune responses elicited and the feasibility of prophylactic vaccines looking forward, will also be discussed.

Review of Requests to Exclude Attenuated Strains of Select Agents and Modified Select Toxins, Division of Select Agents and Toxins, Centers for Disease Control and Prevention, 2003-2017.

The Centers for Disease Control and Prevention's Division of Select Agents and Toxins (DSAT) regulates the possession, use, and transfer of select agents and toxins throughout the United States as part of the Federal Select Agent Program. The Department of Health and Human Services (HHS) select agent regulations also include criteria for the exclusion of select agents and toxins from the requirements of the regulations (42 CFR § 73.3 and 73.4). An entity may request the exclusion of an attenuated strain of a select agent or a select toxin modified to be less potent or toxic. The Intragovernmental Select Agents and Toxins Technical Advisory Committee (ISATTAC) reviews the exclusion request by conducting a risk assessment to determine whether the attenuated strain or modified toxin has the potential to pose a severe risk to public health and safety. In this study, DSAT analyzed the number and types of exclusion requests reviewed by the ISATTAC from January 2003 through December 2017. As of December 2017, DSAT has excluded 50 strains of biological agents and 10 modified toxins from the select agent regulations. The select agent regulations provision for the exclusion of attenuated select agents or modified toxins that no longer have the potential to pose a severe threat to public health and safety is an important mechanism for reducing the regulatory burden on entities that do not need to work with the fully virulent or toxic forms of the agent or toxin. This provision may have the added benefit of encouraging entities to consider working with variants of select agents or toxins that are of less risk than the fully virulent or toxic forms in their research studies and as a positive control.

Effects of periconceptional maternal alcohol intake and a postnatal high-fat diet on obesity and liver disease in male and female rat offspring.

The effects of maternal alcohol consumption around the time of conception on offspring are largely unknown and difficult to determine in a human population. This study utilized a rodent model to examine if periconceptional alcohol (PC:EtOH) consumption, alone or in combination with a postnatal high-fat diet (HFD), resulted in obesity and liver dysfunction. Sprague-Dawley rats were fed a control or an ethanol-containing [12.5% (vol/vol) EtOH] liquid diet from 4 days before mating until 4 days of gestation ( n = 12/group). A subset of offspring was fed a HFD between 3 and 8 mo of age. In males, PC:EtOH and HFD increased total body fat mass ( PPC:EtOH < 0.05, PHFD < 0.0001); in females, only HFD increased fat mass ( PHFD < 0.0001). PC:EtOH increased microvesicular liver steatosis in male, but not female, offspring. Plasma triglycerides, HDL, and cholesterol were increased in PC:EtOH-exposed males ( PPC:EtOH < 0.05), and LDL, cholesterol, and leptin (Lep) were increased in PC:EtOH-exposed females ( PPC:EtOH < 0.05). mRNA levels of Tnf-α and Lep in visceral adipose tissue were increased by PC:EtOH in both sexes ( PPC:EtOH < 0.05), and Il-6 mRNA was increased in males ( PPC:EtOH < 0.05). These findings were associated with reduced expression of microRNA-26a, a known regulator of IL-6 and TNF-α. Alcohol exposure around conception increases obesity risk, alters plasma lipid and leptin profiles, and induces liver steatosis in a sex-specific manner. These programmed phenotypes were similar to those caused by a postnatal HFD, particularly in male offspring. These results have implications for the health of offspring whose mothers consumed alcohol around the time of conception.

Review of Restricted Experiment Requests, Division of Select Agents and Toxins, Centers for Disease Control and Prevention, 2006-2013.

The Centers for Disease Control and Prevention (CDC) Division of Select Agents and Toxins (DSAT) regulates laboratories that possess, use, or transfer select agents and toxins in the United States. DSAT also mitigates biosafety risks through the review of "restricted experiments," which under the select agent regulations are experiments that pose heightened biosafety risks. From January 2006 through December 2013, DSAT received 618 requests from 109 entities to perform potentially restricted experiments. Of these requests, 85% were determined not to meet the regulatory definition of a restricted experiment, while 15% of the requests met the definition of a restricted experiment. Of the 91 restricted experiments proposed, DSAT approved 31 (34%) requests because the biosafety conditions proposed were commensurate with the experiments' biosafety risk. All 31 approved restricted experiments were for work with select toxins. DSAT did not approve 60 restricted experiment requests due to potentially serious biosafety risks to public health and safety. All 60 denied restricted experiments proposed inserting drug resistance traits into select agents that could compromise the control of disease. The select agents and toxins associated most frequently with requests that met the regulatory definition of a restricted experiment are Shiga toxin (n = 16), Burkholderia mallei (n = 15), Botulinum neurotoxin (n = 14), and Brucella abortus (n = 14). In general, all restricted experiment decisions are determined on a case-by-case basis. This article describes the trends and characteristics of the data associated with restricted experiment requests among select agents that have an impact on public health and safety (HHS only agents) or both public health and safety and animal health or products (overlap agents). The information presented here, coupled with the information published in the restricted experiment guidance document ( www.selectagents.gov ), is intended to promote awareness among the research community of the type of experiments that meet the regulatory definition of a restricted experiment as well as to provide a greater understanding of the restricted experiment review process.

Biosafety Recommendations for Work with Influenza Viruses Containing a Hemagglutinin from the A/goose/Guangdong/1/96 Lineage.

The CDC and National Institutes of Health (NIH) Biosafety in Microbiological and Biomedical Laboratories (BMBL) manual describes biosafety recommendations for work involving highly pathogenic avian influenza (HPAI) (US Department of Health and Human Services [HHS], CDC. Biosafety in microbiological and biomedical laboratories, 5th ed. Atlanta, GA: CDC; 2009. HHS publication no. [CDC] 21-1112. Available at http://www.cdc.gov/biosafety/publications/bmbl5). The U.S. Department of Agriculture Guidelines for Avian Influenza Viruses builds on the BMBL manual and provides additional biosafety and biocontainment guidelines for laboratories working with HPAI (US Department of Agriculture, Animal and Plant Health Inspection Service, Agricultural Select Agent Program. Guidelines for avian influenza viruses. Washington, DC: US Department of Agriculture; 2011. Available at http://www.selectagents.gov/Guidelines_for_Avian_Influenza_Viruses.html). The recommendations in this report, which are intended for laboratories in the United States, outline the essential baseline biosafety measures for working with the subset of influenza viruses that contain a hemagglutinin (HA) from the HPAI influenza A/goose/Guangdong/1/96 lineage, including reassortant influenza viruses created in a laboratory setting. All H5N1 influenza virus clades known to infect humans to date have been derived from this lineage (WHO/OIE/FAO H5N1 Evolution Working Group. Continued evolution of highly pathogenic avian influenza A [H5N1]: updated nomenclature. Influenza Other Respir Viruses 2012;6:1-5). In 2009, the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules were amended to include specific biosafety and biocontainment recommendations for laboratories working with Recombinant Risk Group 3 influenza viruses, including HPAI H5N1 influenza viruses within the Goose/Guangdong/1/96-like H5 lineage. In February 2013, the NIH guidelines were further revised to provide additional biosafety containment enhancements and practices for research with HPAI H5N1 viruses that are transmissible among mammals by respiratory droplets (i.e., mammalian-transmissible HPAI H5N1) (National Institutes of Health, Office of Biotechnology Activities. NIH guidelines for research involving recombinant or synthetic nucleic acid molecules. Appendix G-II-C-5: biosafety level 3 enhanced for research involving risk group 3 influenza viruses. Bethesda, MD: National Institutes of Health; 2013. Available at http://oba.od.nih.gov/rdna/nih_guidelines_oba.html). The recent revisions to the NIH guidelines focus on a smaller subset of viruses but are applicable and consistent with the recommendations in this report. The biosafety recommendations in this report were developed by CDC with advice from the Intragovernmental Select Agents and Toxins Technical Advisory Committee, which is a panel composed of federal government subject-matter experts, and from public input received in response to the request for information that was published in the Federal Register on October 17, 2012 (US Department of Health and Human Services, CDC. Influenza viruses containing the hemagglutinin from the Goose/ Guangdong/1/96 lineage; proposed rule; request for information and comment. 42 CFR, Part 73. Federal Register 2012;77:63783-5). Work with HPAI H5N1 virus should be conducted, at a minimum, at biosafety level 3 (BSL-3), with specific enhancements to protect workers, the public, animal health, and animal products. Original clinical specimens suspected of containing viruses of this lineage can only be handled at BSL-2 if the procedures do not involve the propagation of the virus. An appropriate biosafety level should be determined in accordance with a biosafety risk assessment. Additional information on performing biosafety risk assessments and establishing effective biosafety containment is available in the BMBL manual.