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The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA “Animal Rule” for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other “wild type” strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

People infected with the mosquito-borne Rift Valley fever virus (RVFV) can suffer from eye-related problems resulting in ongoing vision issues or even permanent blindness. Despite ocular disease being the most frequently reported severe outcome, it is vastly understudied compared to other disease outcomes caused by RVFV. Ocular manifestations of RVFV include blurred vision, uveitis, and retinitis. When an infected individual develops macular or paramacular lesions, there is a 50% chance of permanent vision loss in one or both eyes. The cause of blinding ocular pathology remains unknown in part due to the lack of a tractable animal model. Using 3 relevant exposure routes, both subcutaneous (SC) and aerosol inoculation of Sprague Dawley rats led to RVFV infection of the eye. Surprisingly, direct inoculation of the conjunctiva did not result in successful ocular infection. The posterior segment of the eye, including the optic nerve, choroid, ciliary body, and retina, were all positive for RVFV antigen in SC-infected rats, and live virus was isolated from the eyes. Proinflammatory cytokines and increased leukocyte counts were also found in the eyes of infected rats. Additionally, human ocular cell lines were permissive for Lrp1-dependent RVFV infection. This study experimentally defines viral tropism of RVFV in the posterior segment of the rat eye and characterizes virally-mediated ocular inflammation, providing a foundation for evaluation of vaccines and therapeutics to protect against adverse ocular outcomes.

Venezuelan equine encephalitis virus (VEEV) remains a risk for epidemic emergence or use as an aerosolized bioweapon. To develop possible countermeasures, we isolated VEEV-specific neutralizing monoclonal antibodies (mAbs) from mice and a human immunized with attenuated VEEV strains. Functional assays and epitope mapping established that potently inhibitory anti-VEEV mAbs bind distinct antigenic sites in the A or B domains of the E2 glycoprotein and block multiple steps in the viral replication cycle including attachment, fusion, and egress. A 3.2-Å cryo-electron microscopy reconstruction of VEEV virus-like particles bound by a human Fab suggests that antibody engagement of the B domain may result in cross-linking of neighboring spikes to prevent conformational requirements for viral fusion. Prophylaxis or postexposure therapy with these mAbs protected mice against lethal aerosol challenge with VEEV. Our study defines functional and structural mechanisms of mAb protection and suggests that multiple antigenic determinants on VEEV can be targeted for vaccine or antibody-based therapeutic development.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals.

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 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, viral 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.

Tularemia is a severe, zoonotic infection caused by the Gram-negative bacterium Francisella tularensis. Inhalation results in a rapid, severe bacterial pneumonia and sepsis, which can be lethal. Because the cynomolgus macaque is the accepted nonhuman primate model for tularemia, we conducted a natural history study of pneumonic tularemia by exposing macaques to target inhaled doses of 50, 500, or 5000 colony forming units (CFU) of F. tularensis subsp. tularensis SCHU S4. Two animals within the 50 CFU group (calculated doses of 10 and 11 CFU) survived the challenge, while the remainder succumbed to infection. Exposure of cynomolgus macaques to aerosolized SCHU S4 resulted in fever, anorexia, increased white blood cell counts, lymphopenia, thrombocytopenia, increased liver enzymes, alterations in electrocardiogram (ECG), and pathological changes typical of infection with F. tularensis, regardless of the challenge dose. Blood pressure dropped during the febrile phase, particularly as temperature began to drop and macaques succumbed to the disease. ECG analysis indicated that in 33% of the macaques, heart rate was not elevated during the febrile phase (Faget’s sign, pulse-temperature disassociation), which has been reported in a similar percentage of human cases. These results indicated that infection of cynomolgus macaques with aerosolized F. tularensis results in similar disease progression and outcome as seen in humans, and that cynomolgus macaques are a reliable animal model to test medical countermeasures against aerosolized F. tularensis.

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 zoonotic emerging Rift Valley fever virus (RVFV) causes sporadic disease in livestock and humans throughout Africa and the Saudi Arabian peninsula. Infection of people with RVFV can occur through mosquito bite or mucosal exposure during butchering or milking of infected livestock. Disease typically presents as a self-limiting fever; however, in rare cases, hepatitis, encephalitis and ocular disease may occur. Recent studies have illuminated the neuropathogenic mechanisms of RVFV in a rat aerosol infection model. Neurological disease in rats is characterized by breakdown of the blood–brain barrier late in infection, infiltration of leukocytes to the central nervous system (CNS) and massive viral replication in the brain. However, the route of RVFV entry into the CNS after inhalational exposure remains unknown. Here, we visualized the entire nasal olfactory route from snout to brain after RVFV infection using RNA in situ hybridization and immunofluorescence microscopy. We found widespread RVFV-infected cells within the olfactory epithelium, across the cribriform plate, and in the glomerular region of the olfactory bulb within 2 days of infection. These results indicate that the olfactory tract is a major route of infection of the brain after inhalational exposure. A better understanding of potential neuroinvasion pathways can support the design of more effective therapeutic regiments for the treatment of neurological disease caused by RVFV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19. ispartof: NATURE vol:586 issue:7830 pages:509-515 ispartof: location:England status: published

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., Author summary SARS-CoV-2 infection can result in asymptomatic, mild or severe disease in humans. Vaccines against SARS-CoV-2 are paramount for combating COVID-19 disease and curtailing the pandemic. Large animal models are critical for early vaccine testing before human clinical trials. Here, we found that infection of African green monkeys (AGM) with SARS-CoV-2 resulted in mild disease yet lesions were detectable by PET/CT imaging of the lungs. Shedding of infectious virus from both respiratory and gastrointestinal tracts was also documented. This study provides a detailed account of the pathogenesis of a low-passage SARS-CoV-2 isolate in the AGM model and suggests that AGM can be used for preclinical evaluation of candidate vaccines and therapeutic interventions.

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-grade18F-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 excellent species to study mild/subclinical COVID-19 disease and have shed light on unknown aspects of long-term virus shedding. They are ideally suited for preclinical evaluation of candidate vaccines and therapeutic interventions.One Sentence SummarySubclinical infection of African green monkeys infected with SARS-CoV-2 results in prolonged shedding of infectious virus from both respiratory and gastrointestinal tracts.

The emergent coronavirus, designated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a zoonotic pathogen that has demonstrated remarkable transmissibility in the human population and is the etiological agent of a current global pandemic called COVID-191. We measured the dynamic (short-term) aerosol efficiencies of SARS-CoV-2 and compared the efficiencies with two other emerging coronaviruses, SARS-CoV (emerged in 2002) and Middle Eastern respiratory syndrome CoV (MERS-CoV; emerged starting in 2012). We also quantified the long-term persistence of SARS-CoV-2 and its ability to maintain infectivity when suspended in aerosols for up to 16 hours.

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.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC50 and EC99) of less than 10 and 100 ng ml-1, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.

Background: Little is known about granuloma progression of Mycobacterium tuberculosis infection in humans. Using serial positron emission tomography and computed tomography (PET/CT) of an animal model that recapitulates human infection with M. tuberculosis, we are able to track lung granulomas. Objective: We characterized the spatial and temporal pattern of granuloma formation during primary infection and reactivation. Methods: Serial PET/CT was performed on cynomolgus macaques (n = 28) during primary and reactivation M. tuberculosis infection. Distances between granulomas during the first six weeks post infection (“primary” granulomas) were compared to new granulomas that developed afterwards (“secondary” granulomas) using nearest neighbor analysis during primary infection, reactivation and between different routes of infection. Results: Secondary granulomas developed within 2 cm of a primary granuloma within the same lung lobe with 80% probability during the course of primary infection, and this same pattern was observed during reactivation of latent infection after immune suppression. Using a logistic growth function, we were able to predict the maximum number of granulomas that would develop over the course of infection with good correlation (R2 = 0.96). Conclusion: These data provide important insights into the dynamic patterns of bacterial dissemination during the earliest phases of primary infection and reactivation tuberculosis. Keywords: Tuberculosis, Macaque, Granuloma, Temporal-spatial, PET/CT

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.

Experimental infection of animals with aerosols containing pathogenic agents is essential for an understanding of the natural history and pathogenesis of infectious disease as well as evaluation of potential treatments. We evaluated whether the Aeroneb nebulizer, a vibrating mesh nebulizer, would serve as an alternative to the Collison nebulizer, the “gold standard” for generating infectious bioaerosols. While the Collison possesses desirable properties that have contributed to its longevity in infectious disease aerobiology, concerns have lingered about the liquid volume and concentration of the infectious agent required to cause disease and the damage that jet nebulization causes to the agent. Fluorescein salt was added to the nebulizer contents to assess pathogen loss during aerosolization. Relative to fluorescein salt, loss of influenza virus during aerosolization was worse with the Collison than with the Aeroneb. Four other viruses also had superior aerosol performance with the Aeroneb. The Aeroneb did not improve the aerosol performance for a vegetative bacterium, Francisella tularensis. Environmental parameters collected during the aerosol challenges indicated that the Aeroneb generated a higher relative humidity in exposure chambers while not affecting other environmental parameters. The aerosol mass median aerodynamic diameter (MMAD) was generally larger and more disperse for aerosols generated by the Aeroneb than what is seen with the Collison, but ≥80% of particles were within the range that would reach the lower respiratory tract and alveolar regions. The improved aerosol performance and generated particle size range suggest that for viral pathogens, the Aeroneb is a suitable alternative to the Collison three-jet nebulizer for use in experimental infection of animals. IMPORTANCE Respiratory infection by pathogens via aerosol remains a major concern for both natural disease transmission as well as intentional release of biological weapons. Critical to understanding the disease course and pathogenesis of inhaled pathogens are studies in animal models conducted under tightly controlled experimental settings, including the inhaled dose. The route of administration, particle size, and dose can affect disease progression and outcome. Damage to or loss of pathogens during aerosolization could increase the dose required to cause disease and could stimulate innate immune responses, altering outcome. Aerosol generators that reduce pathogen loss would be ideal. This study compares two aerosol generators to determine which is superior for animal studies. Aerosol research methods and equipment need to be well characterized to optimize the development of animal models for respiratory pathogens, including bioterrorism agents. This information will be critical for pivotal efficacy studies in animals to evaluate potential vaccines or treatments against these agents.

Inhalation of Francisella tularensis causes pneumonic tularemia in humans, a severe disease with a 30 to 60% mortality rate. The reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing F. tularensis. We evaluated the relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize the spray factor (SF), a central metric of aerosolization. This optimization reduced both inter- and intraday variability and was applicable to multiple isolates of F. tularensis. Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of the bacterial culture optical density and the number of CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Live vaccine strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent F. tularensis SCHU S4 that had been cultivated in either Mueller-Hinton broth (MHB) or brain heart infusion (BHI) broth. LVS-vaccinated animals challenged with SCHU S4 that had been cultivated in MHB experienced short febrile periods (median, 3.2 days), limited weight loss (10%) than the unvaccinated controls and median survival times that were not significantly different from those for the unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents.

Experimental infection of animals via inhalation containing pathogenic agents is essential to understanding the natural history and pathogenesis of infectious disease as well as evaluation of potential medical countermeasures. We evaluated whether the Aeroneb, a vibrating mesh nebulizer, would serve as an alternative to the Collison, the ‘gold standard’ for generating infectious bioaerosols. While the Collison possesses desirable properties that have contributed to its longevity in infectious disease aerobiology, concerns have lingered about the volume and concentration of agent required to cause disease and the damage that jet nebulization causes to the agent. For viruses, the ratio of aerosol concentration to nebulizer concentration (spray factor, SF), the Aeroneb was superior to the Collison for four different viruses in a nonhuman primate head-only exposure chamber. Aerosol concentration of influenza was higher relative to fluorescein for the Aeroneb compared to the Collison, suggesting that the Aeroneb was less harsh to viral pathogens than the Collison when generating aerosols. The Aeroneb did not improve the aerosol SF for a vegetative bacterium,Francisella tularensis.Environmental parameters collected during the aerosols indicated that the Aeroneb generated a higher relative humidity in exposure chambers while not affecting other environmental parameters. Aerosol mass median aerodynamic diameter was generally larger and more disperse for aerosols generated by the Aeroneb than what is seen with the Collison but ≥80% were within the range that would reach the lower respiratory tract and alveolar regions. These data suggest that for viral pathogens, the Aeroneb is a suitable alternative to the Collison 3-jet nebulizer.ImportanceThe threat of aerosolization is often not the natural method of transmission. While selection of an appropriate animal model is vital for these types of experiments, other confounding factors can be controlled through a thorough understanding of experimental design and the effects that different parameters can have on disease outcome. Route of administration, particle size, and dose are all factors which can affect disease progression and need to be controlled. Aerosol research methods and equipment need to be well characterized to optimize the development of animal models for bioterrorism agents.

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 year 2015 is as far away in chronological time from 1965 as 1965 was from World War I. In other words, Lyndon Baines Johnson and ESEA are as remote and distant to our students as Archduke Ferdinand was to us in our school days. What seems to some of us like yesterday, or the start of a recent era, is in fact now a distant and remote time, seemingly unconnected from our present educational predicaments—at least in the eyes of our students. Or, to put it another way, ESEA is now fit for historical study.

Francisella tularensis, a gram negative coccobacillus, is the causative agent of tularemia, a severe zoonotic infection. When inhaled, as few as 15 cfu is sufficient to cause disease. There is concern that tularemia could be used as a biological weapon so vaccines are urgently needed. We have previously shown that the New Zealand White (NZW) rabbit is a good model of human disease; after exposure to small particle aerosols containing virulent F. tularensis at doses as low as 66 cfu, naive NZW rabbits develop fever within 3 days and succumb within 4–7 days. Disease pathology in the NZW rabbit strongly resembles what has been reported in humans. Recombinant derivatives of a virulent strain of F. tularensis are highly attenuated in NZW rabbits and protect against aerosol challenge with virulent F. tularensis. The level of protection is dependent on the strain used, the route of vaccination, and the number of vaccinations. Using a prime-boost small particle aerosol vaccination regimen, 73% percent of rabbits survived a robust (100 LD50) challenge when vaccinated with a strain that is deficient in the aromatic amino acid synthase pathway. Pre-challenge, post-vaccination plasma IgG and IgM titers against heat-killed F. tularensis correlate with protection. A single inoculation of plasma from hyper-immune rabbits extended the survival of naïve rabbits by one day. We will report our efforts to evaluate the role of antibody in protection of NZW rabbits against tularemia, including passive immunization of mice using purified immunoglobulin from hyper-immune rabbits and in vitro assays of antibody function.

Rift Valley fever virus (RVFV) is a zoonotic disease of livestock that causes several clinical outcomes in people including febrile disease, hemorrhagic fever, and/or encephalitis. After aerosol infection with RVFV, Lewis rats develop lethal encephalitic disease, and we use this as a model for studying disease mechanisms of RVFV infection in the brain. Permeability of the brain vasculature in relation to virus invasion and replication is not known. Here, we found that vascular permeability in the brain occurred late in the course of infection and corresponded temporally to expression of matrix metalloproteinase-9 (MMP-9). Virus replication was ongoing within the central nervous system for several days prior to detectable vascular leakage. Based on this study, vascular permeability was not required for entry of RVFV into the brain of rats. Prevention of vascular leakage late in infection may be an important component for prevention of lethal neurological disease in the rat model.

Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections.

The most important piece of education legislation in U.S. history, which had its fiftieth anniversary on April 11, 2015, is a law most people have never heard of. Parents do not discuss it on the sidelines of children's sports events. Teachers do not hear about it in professional development

Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and humans. Although the disease is caused by a mosquito-borne virus, humans are infected through contact with, or inhalation of, virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis, resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus, animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or a sublethal dose of RVFV. Divergence between lethal and sublethal infections occurred as early as 2 days postinfection (dpi), at which point CD8 + T cells from lethally infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of major histocompatibility complex (MHC) class II molecules, in contrast to surviving animals. At 4 dpi, lethally infected animals failed to demonstrate proliferation of total CD4 + and CD8 + T cells, in contrast to survivors. These marked changes in peripheral blood cells occur much earlier than more-established indicators of severe RVF disease, such as granulocytosis and fever. In addition, an early proinflammatory (gamma interferon [IFN-γ], interleukin 6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1]) and antiviral (IFN-α) response was seen in survivors, while very late cytokine expression was found in animals with lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow the testing of therapeutics and vaccines in the AGM model. IMPORTANCE Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF lead to death or significant long-term disability for infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here we report the similarities in disease course between infected AGM and humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus that causes low mortality but high morbidity rates in humans. In addition to natural outbreaks, there is the potential for exposure to VEEV via aerosolized virus particles. There are currently no FDA-licensed vaccines or antiviral therapies for VEEV. Passive immunotherapy is an approved method used to protect individuals against several pathogens and toxins. Human polyclonal antibodies (PAbs) are ideal, but this is dependent upon serum from convalescent human donors, which is in limited supply. Non-human-derived PAbs can have serious immunoreactivity complications, and when “humanized,” these antibodies may exhibit reduced neutralization efficiency. To address these issues, transchromosomic (Tc) bovines have been created, which can produce potent neutralizing human antibodies in response to hyperimmunization. In these studies, we have immunized these bovines with different VEEV immunogens and evaluated the protective efficacy of purified preparations of the resultant human polyclonal antisera against low- and high-dose VEEV challenges. These studies demonstrate that prophylactic or therapeutic administration of the polyclonal antibody preparations (TcPAbs) can protect mice against lethal subcutaneous or aerosol challenge with VEEV. Furthermore, significant protection against unrelated coinfecting viral pathogens can be conferred by combining individual virus-specific TcPAb preparations. IMPORTANCE With the globalization and spread or potential aerosol release of emerging infectious diseases, it will be critical to develop platforms that are able to produce therapeutics in a short time frame. By using a transchromosomic (Tc) bovine platform, it is theoretically possible to produce antigen-specific highly neutralizing therapeutic polyclonal human antibody (TcPAb) preparations in 6 months or less. In this study, we demonstrate that Tc bovine-derived Venezuelan equine encephalitis virus (VEEV)-specific TcPAbs are highly effective against VEEV infection that mimics not only the natural route of infection but also infection via aerosol exposure. Additionally, we show that combinatorial TcPAb preparations can be used to treat coinfections with divergent pathogens, demonstrating that the Tc bovine platform could be beneficial in areas where multiple infectious diseases occur contemporaneously or in the case of multipathogen release.