Structural and functional basis of VLDLR usage by Eastern equine encephalitis virus.

The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

Persistence of Influenza H5N1 and H1N1 Viruses in Unpasteurized Milk on Milking Unit Surfaces.

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.

Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

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-1ß 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.

Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

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.

Advances and gaps in SARS-CoV-2 infection models.

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.

Rift Valley Fever Virus Infects the Posterior Segment of the Eye and Induces Inflammation in a Rat Model of Ocular Disease.

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. IMPORTANCE Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring. Despite the prevalence and severity of this outcome, very little is known about the mechanisms of pathogenesis. We addressed this gap by developing a rodent model of ocular disease. Subcutaneous infection of Sprague Dawley rats resulted in infection of the uvea, retina, and optic nerve along with the induction of inflammation within the posterior eye. Infection of human ocular cells induced inflammatory responses and required host entry factors for RVFV infection similar to rodents. This work provides evidence of how RVFV infects the eye, and this information can be applied to help mitigate the devastating outcomes of RVF ocular disease through vaccines or treatments.

Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques.

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.

SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and shedding of infectious virus from both respiratory and gastrointestinal tracts.

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.

Development of Rift valley fever encephalitis in rats is mediated by early infection of olfactory epithelium and neuroinvasion across the cribriform plate.

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.

Rational design of a live-attenuated eastern equine encephalitis virus vaccine through informed mutation of virulence determinants.

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.

Neutrophil and macrophage influx into the central nervous system are inflammatory components of lethal Rift Valley fever encephalitis in rats.

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.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

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.

Development, Characterization, and Standardization of a Nose-Only Inhalation Exposure System for Exposure of Rabbits to Small-Particle Aerosols Containing Francisella tularensis.

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 (<5%), and longer median survival times (∼18 days) that were significantly different from those for unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with SCHU S4 that had been cultivated in BHI experienced longer febrile periods (median, 5.5 days) and greater 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.

Peripheral Blood Biomarkers of Disease Outcome in a Monkey Model of Rift Valley Fever Encephalitis.

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.

A Vibrating Mesh Nebulizer as an Alternative to the Collison Three-Jet Nebulizer for Infectious Disease Aerobiology.

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.

Widespread Virus Replication in Alveoli Drives Acute Respiratory Distress Syndrome in Aerosolized H5N1 Influenza Infection of Macaques.

Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and IFN-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

Antibody Preparations from Human Transchromosomic Cows Exhibit Prophylactic and Therapeutic Efficacy against Venezuelan Equine Encephalitis Virus.

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.

Aerosolized rift valley fever virus causes fatal encephalitis in african green monkeys and common marmosets.

Rift Valley fever (RVF) is a veterinary and human disease in Africa and the Middle East. The causative agent, RVF virus (RVFV), can be naturally transmitted by mosquito, direct contact, or aerosol. We sought to develop a nonhuman primate (NHP) model of severe RVF in humans to better understand the pathogenesis of RVF and to use for evaluation of medical countermeasures. NHP from four different species were exposed to aerosols containing RVFV. Both cynomolgus and rhesus macaques developed mild fevers after inhalation of RVFV, but no other clinical signs were noted and no macaque succumbed to RVFV infection. In contrast, both marmosets and African green monkeys (AGM) proved susceptible to aerosolized RVF virus. Fever onset was earlier with the marmosets and had a biphasic pattern similar to what has been reported in humans. Beginning around day 8 to day 10 postexposure, clinical signs consistent with encephalitis were noted in both AGM and marmosets; animals of both species succumbed between days 9 and 11 postexposure. Marmosets were susceptible to lower doses of RVFV than AGM. Histological examination confirmed viral meningoencephalitis in both species. Hematological analyses indicated a drop in platelet counts in both AGM and marmosets suggestive of thrombosis, as well as leukocytosis that consisted mostly of granulocytes. Both AGM and marmosets would serve as useful models of aerosol infection with RVFV.

Combined alphavirus replicon particle vaccine induces durable and cross-protective immune responses against equine encephalitis viruses.

Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal. Importance: Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the surface proteins of all three viruses. In this report we demonstrate in both mice and macaques that this combined vaccine is safe, generates a strong immune response, and protects against aerosol challenge with the viruses that cause Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis.

Live attenuated mutants of Francisella tularensis protect rabbits against aerosol challenge with a virulent type A strain.

Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.