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We aerosolized severe acute respiratory syndrome coronavirus 2 and determined that its dynamic aerosol efficiency surpassed those of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome. Although we performed experiment only once across several laboratories, our findings suggest retained infectivity and virion integrity for up to 16 hours in respirable-sized aerosols.

Circoviridae is a family of circular single-stranded DNA viruses whose members infect a wide variety of hosts. While well characterized in avian and mammalian hosts, little is known about circoviruses associated with Antarctic animals. From 48 Weddell seal (Leptonychotes weddellii) fecal samples collected on the sea ice in McMurdo between Nov 2014 and Dec 2014, we identified and determined the genomes of novel viruses that fall within two genera of the family Circoviridae, i.e. Circovirus (n = 7) and Cyclovirus (n = 45). We named these viruses as werosea circovirus (WerCV) and werosea cyclovirus (WerCyV). The genomes of WerCV and WerCyV share ~63–64% genome-wide pairwise identity with classified circoviruses and cycloviruses, respectively. Based on the species demarcation threshold of 80% for members of the Circoviridae, the genomes of WerCV and WerCyV represent new species in their respective genera. Evidence indicated recombination in five of the 45 WerCyV genomes identified in this study. These are the first circoviruses found associated with Antarctic pinnipeds, adding to those recently identified associated with Adelie (Pygoscelis adeliae) and chinstrap penguins (P. antarcticus).

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

Background Swine influenza A virus (IAV) reassortment with 2009 H1N1 pandemic (H1N1pdm09) virus has been documented, and new genotypes and subclusters of H3N2 have since expanded in the US swine population. An H3N2 variant (H3N2v) virus with the H1N1pdm09 matrix gene and the remaining genes of swine triple reassortant H3N2 caused outbreaks at agricultural fairs in 2011–2012. Methods To assess commercial swine IAV vaccines' efficacy against H3N2 viruses, including those similar to H3N2v, antisera to three vaccines were tested by hemagglutinin inhibition (HI) assay against contemporary H3N2. Vaccine 1, with high HI cross-reactivity, was further investigated for efficacy against H3N2 virus infection in pigs with or without maternally derived antibodies (MDA). In addition, efficacy of a vaccine derived from whole inactivated virus (WIV) was compared with live attenuated influenza virus (LAIV) against H3N2. Results Hemagglutinin inhibition cross-reactivity demonstrated that contemporary swine H3N2 viruses have drifted from viruses in current swine IAV vaccines. The vaccine with the highest level of HI cross-reactivity significantly protected pigs without MDA. However, the presence of MDA at vaccination blocked vaccine efficacy. The performance of WIV and LAIV was comparable in the absence of MDA. Conclusions Swine IAV in the United States is complex and dynamic. Vaccination to minimize virus shedding can help limit transmission of virus among pigs and people. However, vaccines must be updated. A critical review of the use of WIV in sows is required in the context of the current IAV ecology and vaccine application in pigs with MDA.

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., Author Summary Broad-spectrum antiviral drugs are preferred because they have the capacity to treat a range of viral illnesses rather than just one. Food and Drug Administration (FDA) approval of antiviral drugs to treat neglected tropical diseases is difficult to obtain due to ethical and logistical considerations when conducting human clinical trials. Rift Valley Fever (RVF) is an endemic tropical disease that causes human morbidity and mortality, as well as economic damage to the livestock industry. There are no licensed antiviral drugs to treat RVF. In this study, we found that a novel anti-influenza drug, Favipiravir (T-705), is able to prevent lethal RVF in rats, and therefore shows promise as a broad-spectrum antiviral treatment.

Recent events surrounding emerging infectious diseases, bioterrorism and increasing multidrug antibiotic resistance in bacteria have drastically increased current needs for effective vaccines. Many years of study have shown that live, attenuated pathogens are often more effective at delivering heterologous protein or DNA to induce protective immune responses. However, these vaccine carriers have inherent safety concerns that have limited their development and their use in many patient populations. Studies using nonliving delivery mechanisms have shown that providing both protein antigen and DNA encoding the antigen to an individual induces an improved, more protective immune response but rarely, if ever, are both delivered simultaneously. Here, non-replicating bacterial minicells derived from a commensal E. coli strain are shown to effectively induce antigen-specific immune responses after simultaneous protein and DNA delivery. These data demonstrate the potential use of achromosomal bacterial minicells as a vaccine carrier.

Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT., Author Summary Leprosy is a progressive disease of the skin and nervous system caused by the bacillus, Mycobacterium leprae. Implementation of multiple drug therapy (MDT) for leprosy has significantly reduced the global cases of leprosy. Currently, only a few endemic countries remain where relatively high number of cases persists. Despite global reduction of leprosy and the concomitant decrease in human reservoirs, leprosy transmission and incidence have not declined as expected, suggesting a possible extra-human or environmental source of the bacilli. In the current study, we demonstrate that M. leprae can survive long-term within cysts of common environmental free-living amoebae. M. leprae residing in amoebal cysts for over 30 days remain fully capable of transferring disease to mouse footpads and retain viability phenotypes after several months residence within amoebal cysts. It is hypothesized that these protozoa provide an intracellular refuge for M. leprae in environments for which they would otherwise seem ill suited. Traits allowing bacilli to survive in macrophages may likely be acquired via an evolutionary response against predation by amoebae. The results from this work suggest alternative non-human reservoirs for M. leprae exist fostering further study to determine the role of amoebae in the transmission of this Mycobacterium to humans.

The lack of identified exposures in 2 of the 11 cases of bioterrorism-related inhalation anthrax in 2001 raised uncertainty about the infectious dose and transmission of Bacillus anthracis. We used the Wells-Riley mathematical model of airborne infection to estimate 1) the exposure concentrations in postal facilities where cases of inhalation anthrax occurred and 2) the risk for infection in various hypothetical scenarios of exposure to B. anthracis aerosolized from contaminated mail in residential settings. These models suggest that a small number of cases of inhalation anthrax can be expected when large numbers of persons are exposed to low concentrations of B. anthracis. The risk for inhalation anthrax is determined not only by bacillary virulence factors but also by infectious aerosol production and removal rates and by host factors.

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

BACKGROUNDThe Canadian National Antiviral Stockpile (NAS) contains treatment for 17.5% of Canadians. This assumes no concurrent intervention strategies and no wastage due to non-influenza respiratory infections. A dynamic model can provide a mechanism to consider complex scenarios to support decisions regarding the optimal NAS size under uncertainty.METHODSWe developed a dynamic model for pandemic influenza in Canada that is structured by age and risk to calculate the demand for antivirals to treat persons with pandemic influenza under a wide-range of scenarios that incorporated transmission dynamics, disease severity, and intervention strategies. The anticipated per capita number of acute respiratory infections due to viruses other than influenza was estimated for the full pandemic period from surveys based on criteria to identify potential respiratory infections.RESULTSOur results demonstrate that up to two thirds of the population could develop respiratory symptoms as a result of infection with a pandemic strain. In the case of perfect antiviral allocation, up to 39.8% of the population could request antiviral treatment. As transmission dynamics, severity and timing of the emergence of a novel influenza strain are unknown, the sensitivity analysis produced considerable variation in potential demand (median: 11%, IQR: 2-21%). If the next pandemic strain emerges in late spring or summer and a vaccine is available before the anticipated fall wave, the median prediction was reduced to 6% and IQR to 0.7-14%. Under the strategy of offering empirical treatment to all patients with influenza like symptoms who present for care, demand could increase to between 65 and 144%.CONCLUSIONSThe demand for antivirals during a pandemic is uncertain. Unless an accurate, timely and cost-effective test is available to identify influenza cases, demand for antivirals from persons infected with other respiratory viruses will be substantial and have a significant impact on the NAS.

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.

Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection., Author Summary The malaria parasite Plasmodium falciparum avoids recognition and clearance by the immune system by sequentially switching between members of the var multi-gene family which encode the immunodominant surface proteins PfEMP1. However, some mechanism must exist to prevent rapid exposure of the pathogen's entire antigenic repertoire as this would quickly terminate the infection. It has previously been shown that the immune system can play an important role in orchestrating the sequential display of variants once an infection is established; however this does not explain how repertoire exhaustion is avoided in the initial phases of infection before an immune response has been established. Here we show that P. falciparum has evolved a highly structured switching pattern to prevent repertoire exhaustion in the early stages of infection without compromising the ability to establish new infections among partially immune individuals.

SUMMARY ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.

In the U.S., despite available swine influenza virus (SIV) vaccines, multiple influenza subtypes as well as antigenic and genetic variants within subtypes continue to circulate in the swine population. One of the challenges to control and eliminate SIV is that the currently used inactivated influenza virus vaccines do not provide adequate cross-protection against multiple antigenic variants of SIV in the field. We previously generated a recombinant H3N2 swine influenza virus (SIV) based on the influenza A/SW/TX/4199-2/98 virus (TX98) containing an NS1 gene expressing a truncated NS1 protein of 126 amino acids, TX98-NS1Delta126 virus. This recombinant strain was demonstrated to be highly attenuated in swine and showed potential for use as a modified live-virus vaccine (MLV) after intratracheal application in pigs. However, this route of inoculation is not practical for vaccination in the field. In the present study, we first compared intramuscular and intranasal routes of application of the MLV, and found that the intranasal route was superior in priming the local (mucosal) immune response. Pigs were then vaccinated via the intranasal route and challenged with wild type homologous TX98 H3N2 virus, with a genetic and antigenic variant H3N2 SIV (influenza A/SW/CO/23619/99 virus, CO99) and a heterosubtypic H1N1 SIV (influenza A/SW/IA/00239/2004 virus, IA04). The intranasally vaccinated pigs were completely protected against homologous challenge. In addition, MLV vaccination provided nearly complete protection against the antigenic H3N2 variant CO99 virus. When challenged with the H1N1 IA04 virus, MLV vaccinated animals displayed reduced fever and virus titers despite minimal reduction in lung lesions. In vaccinated pigs, there was no serologic cross-reactivity by HI assays with the heterologous or heterosubtypic viruses. However, there appeared to be substantial cross-reactivity in antibodies at the mucosal level with the CO99 virus in MLV vaccinated pigs.

The relationship between specific antibody profiles and tuberculosis (TB) state was investigated by measuring serum antibody levels to sixMycobacterium tuberculosisantigens in human subjects grouped into four diagnostic categories: active disease, inactive (past) tuberculosis, latent infection without radiographic chest abnormalities, and infection free. Statistical data analyses showed that the latter two groups were serologically indistinguishable and that active tuberculosis and inactive tuberculosis were characterized by different antibody profiles. Antibodies to the 38-kDa antigen, alanine dehydrogenase, and Rv2626c were associated with active TB, while antibodies to the 16-kDa antigen, ferredoxin A, and ESAT-6 were associated with inactive TB. Thus, the targets of the immune response vary with tuberculosis state. The correlation between bacterial antigen production and infection stage was investigated in mice infected withM. tuberculosisby bacterial transcription profiling. It was found that levels of transcripts encoding the sixM. tuberculosisantigens varied during infection. Together, the data indicate that antigen composition of tubercle bacilli varies with stage of infection and that immunoprofiling can distinguish between tuberculosis states.

The purpose of this study was to compare the killing of Actinobacillus actinomycetemcomitans with that of Capnocytophaga spp. by purified cathepsin G and elastase in vitro. Both were sensitive to killing by purified cathepsin G, but only the Capnocytophaga spp. were killed by elastase. Killing by cathepsin G exhibited logarithmic kinetics, was enhanced slightly by alkaline pH, and was enhanced greatly under hypotonic conditions. Treatment of cathepsin G with diisopropyl fluorophosphate significantly reduced its bactericidal activity against Capnocytophaga spp. but not against Escherichia coli or A. actinomycetemcomitans. The bactericidal effects of cathepsin G against Capnocytophaga sputigena and A. actinomycetemcomitans were inhibited by alpha-1-antichymotrypsin, alpha-1-antitrypsin, and alpha-2-macroglobulin but not by bovine serum albumin. We conclude that (i) cathepsin G kills Capnocytophaga spp. and A. actinomycetemcomitans, (ii) elastase kills Capnocytophaga spp., (iii) the bactericidal activity of cathepsin G is enzyme dependent against Capnocytophaga spp. and enzyme independent against A. actinomycetemcomitans, and (iv) natural plasma antiproteases may control both enzyme-dependent and enzyme-independent bactericidal activities of cathepsin G.