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Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the central nervous system (CNS), were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, VEEV genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1β and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age.

Aerosol exposure to eastern equine encephalitis virus (EEEV) can trigger a lethal viral encephalitis in cynomolgus macaques which resembles severe human disease. Biomarkers indicative of central nervous system (CNS) infection by the virus and lethal outcome of disease would be useful in evaluating potential medical countermeasures, especially for therapeutic compounds. To meet requirements of the Animal Rule, a better understanding of the pathophysiology of EEEV-mediated disease in cynomolgus macaques is needed. In this study, macaques given a lethal dose of clone-derived EEEV strain V105 developed a fever between 2-3 days post infection (dpi) and succumbed to the disease by 6 dpi. At the peak of the febrile phase, there was a significant increase in the delta electroencephalography (EEG) power band associated with deep sleep as well as a sharp rise in intracranial pressure (ICP). Viremia peaked early after infection and was largely absent by the onset of fever. Granulocytosis and elevated plasma levels of IP-10 were found early after infection. At necropsy, there was a one hundred- to one thousand-fold increase in expression of traumatic brain injury genes (LIF, MMP-9) as well as inflammatory cytokines and chemokines (IFN-γ, IP-10, MCP-1, IL-8, IL-6) in the brain tissues. Phenotypic analysis of leukocytes entering the brain identified cells as primarily lymphoid (T, B, NK cells) with lower levels of infiltrating macrophages and activated microglia. Massive amounts of infectious virus were found in the brains of lethally-infected macaques. While no infectious virus was found in surviving macaques, quantitative PCR did find evidence of viral genomes in the brains of several survivors. These data are consistent with an overwhelming viral infection in the CNS coupled with a tremendous inflammatory response to the infection that may contribute to the disease outcome. Physiological monitoring of EEG and ICP represent novel methods for assessing efficacy of vaccines or therapeutics in the cynomolgus macaque model of EEEV encephalitis.

Vaccines are urgently needed to combat the global coronavirus disease 2019 (COVID-19) pandemic, and testing of candidate vaccines in an appropriate non-human primate (NHP) model is a critical step in the process. Infection of African green monkeys (AGM) with a low passage human isolate of SARS-CoV-2 by aerosol or mucosal exposure resulted in mild clinical infection with a transient decrease in lung tidal volume. Imaging with human clinical-grade 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) co-registered with computed tomography (CT) revealed pulmonary lesions at 4 days post-infection (dpi) that resolved over time. Infectious virus was shed from both respiratory and gastrointestinal (GI) tracts in all animals in a biphasic manner, first between 2-7 dpi followed by a recrudescence at 14-21 dpi. Viral RNA (vRNA) was found throughout both respiratory and gastrointestinal systems at necropsy with higher levels of vRNA found within the GI tract tissues. All animals seroconverted simultaneously for IgM and IgG, which has also been documented in human COVID-19 cases. Young AGM represent an species to study mild/subclinical COVID-19 disease and with possible insights into live virus shedding. Future vaccine evaluation can be performed in AGM with correlates of efficacy being lung lesions by PET/CT, virus shedding, and tissue viral load.

Alphaviruses such as Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) are arboviruses that can cause severe zoonotic disease in humans. Both VEEV and EEEV are highly infectious when aerosolized and can be used as biological weapons. Vaccines and therapeutics are urgently needed, but efficacy determination requires animal models. The cynomolgus macaque (Macaca fascicularis) provides a relevant model of human disease, but questions remain whether vaccines or therapeutics can mitigate CNS infection or disease in this model. The documentation of alphavirus encephalitis in animals relies on traditional physiological biomarkers and behavioral/neurological observations by veterinary staff; quantitative measurements such as electroencephalography (EEG) and intracranial pressure (ICP) can recapitulate underlying encephalitic processes. We detail a telemetry implantation method suitable for continuous monitoring of both EEG and ICP in awake macaques, as well as methods for collection and analysis of such data. We sought to evaluate whether changes in EEG/ICP suggestive of CNS penetration by virus would be seen after aerosol exposure of naïve macaques to VEEV IC INH9813 or EEEV V105 strains compared to mock-infection in a cohort of twelve adult cynomolgus macaques. Data collection ran continuously from at least four days preceding aerosol exposure and up to 50 days thereafter. EEG signals were processed into frequency spectrum bands (delta: [0.4 - 4Hz); theta: [4 - 8Hz); alpha: [8-12Hz); beta: [12-30] Hz) and assessed for viral encephalitis-associated changes against robust background circadian variation while ICP data was assessed for signal fidelity, circadian variability, and for meaningful differences during encephalitis. Results indicated differences in delta, alpha, and beta band magnitude in infected macaques, disrupted circadian rhythm, and proportional increases in ICP in response to alphavirus infection. This novel enhancement of the cynomolgus macaque model offers utility for timely determination of onset, severity, and resolution of encephalitic disease and for the evaluation of vaccine and therapeutic candidates.

Rift Valley fever virus (RVFV) causes severe disease in livestock concurrent with zoonotic transmission to humans. A subset of people infected with RVFV develop encephalitis, and significant gaps remain in our knowledge of how RVFV causes pathology in the brain. We previously found that, in Lewis rats, subcutaneous inoculation with RVFV resulted in subclinical disease while inhalation of RVFV in a small particle aerosol caused fatal encephalitis. Here, we compared the disease course of RVFV in Lewis rats after each different route of inoculation in order to understand more about pathogenic mechanisms of fatal RVFV encephalitis. In aerosol-infected rats with lethal encephalitis, neutrophils and macrophages were the major cell types infiltrating the CNS, and this was concomitant with microglia activation and extensive cytokine inflammation. Despite this, prevention of neutrophil infiltration into the brain did not ameliorate disease. Unexpectedly, in subcutaneously-inoculated rats with subclinical disease, detectable viral RNA was found in the brain along with T-cell infiltration. This study sheds new light on the pathogenic mechanisms of RVFV encephalitis.

Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses.

Tularemia, also known as rabbit fever, is a severe zoonotic disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). While there have been a number of attempts to develop a vaccine for Ft, few candidates have advanced beyond experiments in inbred mice. We report here that a prime-boost strategy with aerosol delivery of recombinant live attenuated candidate Ft S4ΔaroD offers significant protection (83% survival) in an outbred animal model, New Zealand White rabbits, against aerosol challenge with 248 cfu (11 LD50) of virulent type A Ft SCHU S4. Surviving rabbits given two doses of the attenuated strains by aerosol did not exhibit substantial post-challenge fevers, changes in erythrocyte sedimentation rate or in complete blood counts. At a higher challenge dose (3,186 cfu; 139 LD50), protection was still good with 66% of S4ΔaroD-vaccinated rabbits surviving while 50% of S4ΔguaBA vaccinated rabbits also survived challenge. Pre-challenge plasma IgG titers against Ft SCHU S4 corresponded with survival time after challenge. Western blot analysis found that plasma antibody shifted from predominantly targeting Ft O-antigen after the prime vaccination to other antigens after the boost. These results demonstrate the superior protection conferred by a live attenuated derivative of virulent F. tularensis, particularly when given in an aerosol prime-boost regimen.

Whole-brain imaging is becoming a fundamental means of experimental insight; however, achieving subcellular resolution imagery in a reasonable time window has not been possible. We describe the first application of multicolor ribbon scanning confocal methods to collect high-resolution volume images of chemically cleared brains. We demonstrate that ribbon scanning collects images over ten times faster than conventional high speed confocal systems but with equivalent spectral and spatial resolution. Further, using this technology, we reconstruct large volumes of mouse brain infected with encephalitic alphaviruses and demonstrate that regions of the brain with abundant viral replication were inaccessible to vascular perfusion. This reveals that the destruction or collapse of large regions of brain micro vasculature may contribute to the severe disease caused by Venezuelan equine encephalitis virus. Visualization of this fundamental impact of infection would not be possible without sampling at subcellular resolution within large brain volumes.

Rift Valley Fever (RVF) is an emerging viral disease that causes significant human and veterinary illness in Africa and the Arabian Peninsula. Encephalitis is one of the severe complications arising from RVF virus (RVFV) infection of people, and the pathogenesis of this form of RVF is completely unknown. We use a novel reproducible encephalitic disease model in rats to identify biomarkers of lethal infection. Lewis rats were infected with RVFV strain ZH501 by aerosol exposure, then sacrificed daily to determine the course of infection and evaluation of clinical, virological, and immunological parameters. Weight loss, fever, and clinical signs occurred during the last 1 – 2 days prior to death. Prior to onset of clinical indications of disease, rats displayed marked granulocytosis and thrombocytopenia. In addition, high levels of inflammatory chemokines (MCP-1, MCS-F, Gro/KC, RANTES, and IL-1β) were detected first in serum (3-5 dpi) followed by brain (5-7 dpi). The results of this study are consistent with clinical data from human RVF patients and validate Lewis rats as an appropriate small animal model for RVF encephalitis. The biomarkers we identified here will be useful in future studies evaluating the efficacy of novel vaccines and therapeutics.

BACKGROUND: Development of antiviral drugs that have broad-spectrum activity against a number of viral infections would be of significant benefit. Due to the evolution of resistance to currently licensed antiviral drugs, development of novel anti-influenza drugs is in progress, including Favipiravir (T-705), which is currently in human clinical trials. T-705 displays broad-spectrum in vitro activity against a number of viruses, including Rift Valley Fever virus (RVFV). RVF is an important neglected tropical disease that causes human, agricultural, and economic losses in endemic regions. RVF has the capacity to emerge in new locations and also presents a potential bioterrorism threat. In the current study, the in vivo efficacy of T-705 was evaluated in Wistar-Furth rats infected with the virulent ZH501 strain of RVFV by the aerosol route. METHODOLOGY/PRINCIPAL FINDINGS: Wistar-Furth rats are highly susceptible to a rapidly lethal disease after parenteral or inhalational exposure to the pathogenic ZH501 strain of RVFV. In the current study, two experiments were performed: a dose-determination study and a delayed-treatment study. In both experiments, all untreated control rats succumbed to disease. Out of 72 total rats infected with RVFV and treated with T-705, only 6 succumbed to disease. The remaining 66 rats (92%) survived lethal infection with no significant weight loss or fever. The 6 treated rats that succumbed survived significantly longer before succumbing to encephalitic disease. CONCLUSIONS/SIGNIFICANCE: Currently, there are no licensed antiviral drugs for treating RVF. Here, T-705 showed remarkable efficacy in a highly lethal rat model of Rift Valley Fever, even when given up to 48 hours post-infection. This is the first study to show protection of rats infected with the pathogenic ZH501 strain of RVFV. Our data suggest that T-705 has potential to be a broad-spectrum antiviral drug.

In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller-Hinton broth (MHb) or Chamberlain’s Chemically Defined Medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 minutes for BHI compared to

Humans infected with Rift Valley Fever virus (RVFV) generally recover after a febrile illness; however, a proportion of patients progress to a more severe clinical outcome such as hemorrhagic fever or meningoencephalitis. RVFV is naturally transmitted to livestock and humans by mosquito bites, but it is also infectious through inhalational exposure, making it a potential bioterror weapon. To better understand the disease caused by inhalation of RVFV, Wistar-Furth, ACI, or Lewis rats were exposed to experimental aerosols containing virulent RVFV. Wistar-Furth rats developed a rapidly progressing lethal hepatic disease after inhalational exposure; ACI rats were 100-fold less susceptible and developed fatal encephalitis after infection. Lewis rats, which do not succumb to parenteral inoculation with RVFV, developed fatal encephalitis after aerosol infection. RVFV was found in the liver, lung, spleen, heart, kidney and brain of Wistar Furth rats that succumbed after aerosol exposure. In contrast, RVFV was found only in the brains of ACI or Lewis rats that succumbed after aerosol exposure. Lewis rats that survived s.c. infection were not protected against subsequent re-challenge by aerosol exposure to the homologous virus. This is the first side-by-side comparison of the lethality and pathogenesis of RVFV in 3 rat strains after aerosol exposure and the first step towards developing a rodent model suitable for use under the FDA Animal Rule to test potential vaccines and therapeutics for aerosol exposure to RVFV.

Pneumonic tularemia is caused by inhalation of the gram negative bacterium, Francisella tularensis. Because of concerns that tularemia could be used as a bioterrorism agent, vaccines and therapeutics are urgently needed. Animal models of pneumonic tularemia with a pathophysiology similar to the human disease are needed to evaluate the efficacy of these potential medical countermeasures.Rabbits exposed to aerosols containing Francisella tularensis strain SCHU S4 developed a rapidly progressive fatal pneumonic disease. Clinical signs became evident on the third day after exposure with development of a fever (>40.5°C) and a sharp decline in both food and water intake. Blood samples collected on day 4 found lymphopenia and a decrease in platelet counts coupled with elevations in erythrocyte sedimentation rate, alanine aminotransferase, cholesterol, granulocytes and monocytes. Radiographs demonstrated the development of pneumonia and abnormalities of intestinal gas consistent with ileus. On average, rabbits were moribund 5.1 days after exposure; no rabbits survived exposure at any dose (190-54,000 cfu). Gross evaluation of tissues taken at necropsy showed evidence of pathology in the lungs, spleen, liver, kidney and intestines. Bacterial counts confirmed bacterial dissemination from the lungs to the liver and spleen.The pathophysiology of pneumonic tularemia in rabbits resembles what has been reported for humans. Rabbits therefore are a relevant model of the human disease caused by type A strains of F. tularensis.

Abstract An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.

Examining the persistence of highly pathogenic avian influenza A(H5N1) from cattle and human influenza A(H1N1)pdm09 pandemic viruses in unpasteurized milk revealed that both remain infectious on milking equipment materials for several hours. Those findings highlight the risk for H5N1 virus transmission to humans from contaminated surfaces during the milking process.

Abstract Although zinc deficiency (secondary to malnutrition) has long been considered an important contributor to morbidity and mortality of infectious disease (e.g. diarrhea disorders), epidemiologic data (including randomized controlled trials with supplemental zinc) for such a role in lower respiratory tract infection are somewhat ambiguous. In the current study, we provide the first preclinical evidence demonstrating that although diet‐induced acute zinc deficiency (Zn‐D: ~50% decrease) did not worsen infection induced by either influenza A (H1N1) or methicillin‐resistant staph aureus (MRSA), Zn‐D mice were sensitive to the injurious effects of superinfection of H1N1 with MRSA. Although the mechanism underlying the sensitivity of ZnD mice to combined H1N1/MRSA infection is unclear, it was noteworthy that this combination exacerbated lung injury as shown by lung epithelial injury markers (increased BAL protein) and decreased genes related to epithelial integrity in Zn‐D mice (surfactant protein C and secretoglobins family 1A member 1). As bacterial pneumonia accounts for 25%–50% of morbidity and mortality from influenza A infection, zinc deficiency may be an important pathology component of respiratory tract infections.

Summary: Highly pathogenic avian influenza A H5N1 viruses cause high mortality in humans and have pandemic potential. Effective vaccines and treatments against this threat are urgently needed. Here, we have refined our previously established model of lethal H5N1 infection in cynomolgus macaques. An inhaled aerosol virus dose of 5.1 log10 plaque-forming unit (pfu) induced a strong febrile response and acute respiratory disease, with four out of six macaques succumbing after challenge. Vaccination with three doses of adjuvanted seasonal quadrivalent influenza vaccine elicited low but detectable neutralizing antibody to H5N1. All six vaccinated macaques survived four times the 50% lethal dose of aerosolized H5N1, while four of six unvaccinated controls succumbed to disease. Although vaccination did not protect against severe influenza, vaccinees had reduced respiratory dysfunction and lower viral load in airways compared to controls. We anticipate that our macaque model will play a vital role in evaluating vaccines and antivirals against influenza pandemics.

In his dissent in Plessy v. Ferguson, Justice John Marshall Harlan wrote, “Our constitution is color-blind, and neither knows nor tolerates classes among citizens.” Racial conservatives have argued that Harlan’s dissent should invalidate policies that partially redress the historical injuries inflicted on African Americans. This article contends that the concept of colorblindness misstates Harlan’s central claim. His dissent insisted black Americans are entitled to the full fruits of citizenship. This rearticulation reveals that race-conscious policies, particularly within public education, fulfill, rather than undermine, Harlan’s primary objective of defending black citizenship. The article shows that racial conservatives’ colorblind ideology emerged as a strategic response to their legal defeats over segregated schools and to attack affirmative action. The Supreme Court’s later use of colorblind principles is contrasted with empirical evidence of the efficacy of color-conscious pedagogies within schools. The recent Gary B. v. Whitmer case highlights the linkages between full citizenship and race-conscious pedagogies.

Pneumonic tularemia is a highly debilitating and potentially fatal disease caused by inhalation of Francisella tularensis. Most of our current understanding of its pathogenesis is based on the highly virulent F. tularensis subsp. tularensis strain SCHU S4. However, multiple sources of SCHU S4 have been maintained and propagated independently over the years, potentially generating genetic variants with altered virulence. In this study, the virulence of four SCHU S4 stocks (NR-10492, NR-28534, NR-643 from BEI Resources and FTS-635 from Battelle Memorial Institute) along with another virulent subsp. tularensis strain, MA00-2987, were assessed in parallel. In the Fischer 344 rat model of pneumonic tularemia, NR-643 and FTS-635 were found to be highly attenuated compared to NR-10492, NR-28534, and MA00-2987. In the NZW rabbit model of pneumonic tularemia, NR-643 caused morbidity but not mortality even at a dose equivalent to 500x the LD50 for NR-10492. Genetic analyses revealed that NR-10492 and NR-28534 were identical to each other, and nearly identical to the reference SCHU S4 sequence. NR-643 and FTS-635 were identical to each other but were found to have nine regions of difference in the genomic sequence when compared to the published reference SCHU S4 sequence. Given the genetic differences and decreased virulence, NR-643/FTS-635 should be clearly designated as a separate SCHU S4 substrain and no longer utilized in efficacy studies to evaluate potential vaccines and therapeutics against tularemia.

The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

Eastern (EEEV) and Venezuelan (VEEV) equine encephalitis viruses (EEVs) are related, (+) ssRNA arboviruses that can cause severe, sometimes fatal, encephalitis in humans. EEVs are highly infectious when aerosolized, raising concerns for potential use as biological weapons. No licensed medical countermeasures exist; given the severity/rarity of natural EEV infections, efficacy studies require animal models. Cynomolgus macaques exposed to EEV aerosols develop fever, encephalitis, and other clinical signs similar to humans. Fever is nonspecific for encephalitis in macaques. Electrocardiography (ECG) metrics may predict onset, severity, or outcome of EEV-attributable disease. Macaques were implanted with thermometry/ECG radiotransmitters and exposed to aerosolized EEV. Data was collected continuously, and repeated-measures ANOVA and frequency-spectrum analyses identified differences between courses of illness and between pre-exposure and post-exposure states. EEEV-infected macaques manifested widened QRS-intervals in severely ill subjects post-exposure. Moreover, QT-intervals and RR-intervals decreased during the febrile period. VEEV-infected macaques suffered decreased QT-intervals and RR-intervals with fever onset. Frequency-spectrum analyses revealed differences in the fundamental frequencies of multiple metrics in the post-exposure and febrile periods compared to baseline and confirmed circadian dysfunction. Heart rate variability (HRV) analyses revealed diminished variability post-exposure. These analyses support using ECG data alongside fever and clinical laboratory findings for evaluating medical countermeasure efficacy.