Prevalence of Antimicrobial Resistance in Select Bacteria From Retail Seafood-United States, 2019.

In 2019, the United States National Antimicrobial Resistance Monitoring System (NARMS) surveyed raw salmon, shrimp, and tilapia from retail grocery outlets in eight states to assess the prevalence of bacterial contamination and antimicrobial resistance (AMR) in the isolates. Prevalence of the targeted bacterial genera ranged among the commodities: Salmonella (0%-0.4%), Aeromonas (19%-26%), Vibrio (7%-43%), Pseudomonas aeruginosa (0.8%-2.3%), Staphylococcus (23%-30%), and Enterococcus (39%-66%). Shrimp had the highest odds (OR: 2.8, CI: 2.0-3.9) of being contaminated with at least one species of these bacteria, as were seafood sourced from Asia vs. North America (OR: 2.7; CI: 1.8-4.7) and Latin America and the Caribbean vs. North America (OR: 1.6; CI: 1.1-2.3) and seafood sold at the counter vs. sold frozen (OR: 2.1; CI: 1.6-2.9). Isolates exhibited pan-susceptibility (Salmonella and P. aeruginosa) or low prevalence of resistance (<10%) to most antimicrobials tested, with few exceptions. Seafood marketed as farm-raised had lower odds of contamination with antimicrobial resistant bacteria compared to wild-caught seafood (OR: 0.4, CI: 0.2-0.7). Antimicrobial resistance genes (ARGs) were detected for various classes of medically important antimicrobials. Clinically relevant ARGs included carbapenemases (bla IMI-2, bla NDM-1) and extended spectrum ß-lactamases (ESBLs; bla CTX-M-55). This population-scale study of AMR in seafood sold in the United States provided the basis for NARMS seafood monitoring, which began in 2020.

Salmonella enterica Serovar Typhimurium Isolates from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type.

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCE Within-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the United States. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

Low occurrence of multi-antimicrobial and heavy metal resistance in Salmonella enterica from wild birds in the United States.

Wild birds are common reservoirs of Salmonella enterica. Wild birds carrying resistant S. enterica may pose a risk to public health as they can spread the resistant bacteria across large spatial scales within a short time. Here, we whole-genome sequenced 375 S. enterica strains from wild birds collected in 41 U.S. states during 1978-2019 to examine bacterial resistance to antibiotics and heavy metals. We found that Typhimurium was the dominant S. enterica serovar, accounting for 68.3% (256/375) of the bird isolates. Furthermore, the proportions of the isolates identified as multi-antimicrobial resistant (multi-AMR: resistant to at least three antimicrobial classes) or multi-heavy metal resistant (multi-HMR: resistant to at least three heavy metals) were both 1.87% (7/375). Interestingly, all the multi-resistant S. enterica (n = 12) were isolated from water birds or raptors; none of them was isolated from songbirds. Plasmid profiling demonstrated that 75% (9/12) of the multi-resistant strains carried resistance plasmids. Our study indicates that wild birds do not serve as important reservoirs of multi-resistant S. enterica strains. Nonetheless, continuous surveillance for bacterial resistance in wild birds is necessary because the multi-resistant isolates identified in this study also showed close genetic relatedness with those from humans and domestic animals.

The Natural History of Aerosolized Francisella tularensis Infection in Cynomolgus Macaques.

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.

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing.

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

Antimicrobial Resistance in Campylobacter Species: Mechanisms and Genomic Epidemiology.

The Campylobacter genus is a large and diverse group of Gram-negative bacteria that are known to colonize humans and other mammals, birds, reptiles, and shellfish. While it is now recognized that several emerging Campylobacter species can be associated with human disease, two species, C. jejuni and C. coli, are responsible for the vast majority of bacterial gastroenteritis in humans worldwide. Infection with C. jejuni, in particular, has also been associated with a number of extragastrointestinal manifestations and autoimmune conditions, most notably Guillain-Barré syndrome. The antimicrobial drugs of choice for the treatment of severe Campylobacter infection include macrolides, such as erythromycin, clarithromycin, or azithromycin. Fluoroquinolones, such as ciprofloxacin, are also commonly used for empirical treatment of undiagnosed diarrheal disease. However, resistance to these and other classes of antimicrobial drugs is increasing and is a major public health problem. The US Centers for Disease Control and Prevention estimates that over 300,000 infections per year are caused by drug-resistant Campylobacter. In this chapter, we discuss the taxonomy of the Campylobacter genus, the clinical and global epidemiological aspects of Campylobacter infection, with an emphasis on C. jejuni and C. coli, and issues related to the treatment of infection and antimicrobial resistance mechanisms. We further discuss the use of next-generation sequencing for the detection and surveillance of antimicrobial resistance genes.

Presence and Persistence of Salmonella in Water: The Impact on Microbial Quality of Water and Food Safety.

Salmonella ranks high among the pathogens causing foodborne disease outbreaks. According to the Centers for Disease Control and Prevention, Salmonella contributed to about 53.4% of all foodborne disease outbreaks from 2006 to 2017, and approximately 32.7% of these foodborne Salmonella outbreaks were associated with consumption of produce. Trace-back investigations have suggested that irrigation water may be a source of Salmonella contamination of produce and a vehicle for transmission. Presence and persistence of Salmonella have been reported in surface waters such as rivers, lakes, and ponds, while ground water in general offers better microbial quality for irrigation. To date, culture methods are still the gold standard for detection, isolation and identification of Salmonella in foods and water. In addition to culture, other methods for the detection of Salmonella in water include most probable number, immunoassay, and PCR. The U.S. Food and Drug Administration (FDA) issued the Produce Safety Rule (PSR) in January 2013 based on the Food Safety Modernization Act (FSMA), which calls for more efforts toward enhancing and improving approaches for the prevention of foodborne outbreaks. In the PSR, agricultural water is defined as water used for in a way that is intended to, or likely to, contact covered produce, such as spray, wash, or irrigation. In summary, Salmonella is frequently present in surface water, an important source of water for irrigation. An increasing evidence indicates irrigation water as a source (or a vehicle) for transmission of Salmonella. This pathogen can survive in aquatic environments by a number of mechanisms, including entry into the viable but nonculturable (VBNC) state and/or residing within free-living protozoa. As such, assurance of microbial quality of irrigation water is critical to curtail the produce-related foodborne outbreaks and thus enhance the food safety. In this review, we will discuss the presence and persistence of Salmonella in water and the mechanisms Salmonella uses to persist in the aquatic environment, particularly irrigation water, to better understand the impact on the microbial quality of water and food safety due to the presence of Salmonella in the water environment.

Draft Genome Sequences of Bifidobacterium Strains Isolated from Dietary Supplements and Cultured Food Products.

Here, we present the genome sequences of 23 Bifidobacterium isolates from several commercially available dietary supplements and cultured food products. Strains of this genus are natural inhabitants of the mammalian mouth, gastrointestinal tract, and vagina. Some species are considered beneficial to human health.

Fifty-Six Draft Genome Sequences of 10 Lactobacillus Species from 22 Commercial Dietary Supplements.

Here, we present the genome sequences of 56 isolates of 10 species of the genus Lactobacillus that are considered beneficial components of the gut microbiota. The isolates examined were found in commercially available dietary supplements in the U.S. market.

Use of whole-genome sequencing for Campylobacter surveillance from NARMS retail poultry in the United States in 2015.

Whole genome sequencing (WGS) has become a rapid and affordable tool for public health surveillance and outbreak detection. In this study, we used the Illuminia MiSeq® to sequence 589 Campylobacter isolates obtained in 2015 from retail poultry meats as part of the National Antimicrobial Resistance Monitoring System (NARMS). WGS data were used to identify the Campylobacter species and to compare the concordance between resistance genotypes and phenotypes. WGS accurately identified 386 C. jejuni and 203 C. coli using gyrA sequence information. Ten resistance genes, including tetO, blaOXA-61, aph(2″)-Ic, aph(2″)-If, aph(2″)-Ig, aph(3')-III, ant(6)-1a, aadE, aph(3")-VIIa, and Inu(C), plus mutations in housekeeping genes (gyrA at position 86, 23S rRNA at position 2074 and 2075), were identified by WGS analysis. Overall, there was a high concordance between phenotypic resistance to a given drug and the presence of known resistance genes. Concordance between both resistance and susceptible phenotypes and genotype was 100% for ciprofloxacin, nalidixic acid, gentamicin, azithromycin, and florfenicol. A few discrepancies were observed for tetracycline, clindamycin, and telithromycin. The concordance between resistance phenotype and genotype ranged from 67.9% to 100%; whereas, the concordance between susceptible phenotype and genotype ranged from 98.0% to 99.6%. Our study demonstrates that WGS can correctly identify Campylobacter species and predict antimicrobial resistance with a high degree of accuracy.

Gyrase A Mutations in Campylobacter Associated with Decreased Susceptibility to Different Fluoroquinolones.

Campylobacter is a leading cause of foodborne diarrheal illness worldwide, and the emergence of antimicrobial-resistant strains is a major global public health concern. The goal of this study was to compare the activity of different fluoroquinolone antibiotics against ciprofloxacin-resistant Campylobacter jejuni and Campylobacter coli. Isolates from retail meats collected between 2002 and 2009 were selected based on their in vitro susceptibility testing results against ciprofloxacin. In total, 289 C. jejuni and 165 C. coli were collected and analyzed. All ciprofloxacin-resistant isolates had a single mutation (Thr86Ile) in their gyrase A (gyrA) gene and exhibited decreased susceptibility to all eight fluoroquinolones tested. Gatifloxacin, enrofloxacin, and levofloxacin showed greater activity than the other fluoroquinolone drugs in both ciprofloxacin-sensitive and -resistant strains.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the ßTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of ßTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques.

Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83-100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI.

Occurrence and Diversity of Clinically Important Vibrio Species in the Aquatic Environment of Georgia.

Among the more than 70 different Vibrio species inhabiting marine, estuarine, and freshwater ecosystems, 12 are recognized as human pathogens. The warm subtropical climate of the Black Sea coastal area and inland regions of Georgia likely provides a favorable environment for various Vibrio species. From 2006 to 2009, the abundance, ecology, and diversity of clinically important Vibrio species were studied in different locations in Georgia and across seasons. Over a 33-month period, 1,595 presumptive Vibrio isolates were collected from the Black Sea (n = 657) and freshwater lakes around Tbilisi (n = 938). Screening of a subset of 440 concentrated and enriched water samples by PCR-electrospray ionization/mass spectrometry (PCR-ESI/MS) detected the presence of DNA from eight clinically important Vibrio species: V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus, V. harveyi, V. metschnikovii, and V. cincinnatiensis. Almost 90% of PCR/ESI-MS samples positive for Vibrio species were collected from June through November. Three important human-pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, and V. vulnificus) were detected in 62.8, 37.8, and 21.4% of samples testing positive for Vibrios, respectively. The results of these activities suggest that natural reservoirs for human-pathogenic Vibrios exist in Georgian aquatic environments. Water temperature at all sampling sites was positively correlated with the abundance of clinically important Vibrio spp. (except V. metschnikovii), and salinity was correlated with species composition at particular Black Sea sites as well as inland reservoirs.

Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

BACKGROUND: Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. METHODS: We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. RESULTS: All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. CONCLUSIONS: We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques.

United States FDA's emergency use authorization of Ebola virus diagnostics: current impact and lessons for the future.

The Ebola outbreak that took hold in West Africa in 2014 outran the epidemic response capacity of many organizations. Five months after the epidemic was first declared, there were still only two laboratories in West Africa with the capacity to confirm Ebola virus infection. In the summer of 2014, before the first case of imported Ebola occurred in the USA, the US FDA announced it would issue Emergency Use Authorizations for Ebola virus in vitro diagnostics to speed their availability. Between October 2014 and March 2015, the FDA issued Emergency Use Authorizations for nine diagnostic products. The actions of the FDA not only allowed nationwide deployment of Ebola virus testing capacity in the USA but also established an attractive regulatory goalpost for companies developing assays for use in West Africa. Here, we comment on the diagnostic assays for which the FDA has issued emergency authorizations and their fitness for purpose.

Methods for Detecting the Environmental Coccoid Form of Helicobacter pylori.

Helicobacter pylori is recognized as the most common pathogen to cause gastritis, peptic and duodenal ulcers, and gastric cancer. The organisms are found in two forms: (1) spiral-shaped bacillus and (2) coccoid. H. pylori coccoid form, generally found in the environment, is the transformed form of the normal spiral-shaped bacillus after exposed to water or adverse environmental conditions such as exposure to sub-inhibitory concentrations of antimicrobial agents. The putative infectious capability and the viability of H. pylori under environmental conditions are controversial. This disagreement is partially due to the fact of lack in detecting the coccoid form of H. pylori in the environment. Accurate and effective detection methods of H. pylori will lead to rapid treatment and disinfection, and less human health damages and reduction in health care costs. In this review, we provide a brief introduction to H. pylori environmental coccoid forms, their transmission, and detection methods. We further discuss the use of these detection methods including their accuracy and efficiency.

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health.

Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

Pre-symptomatic diagnosis and treatment of filovirus diseases.

Filoviruses are virulent human pathogens which cause severe illness with high case fatality rates and for which there are no available FDA-approved vaccines or therapeutics. Diagnostic tools including antibody- and molecular-based assays, mass spectrometry, and next-generation sequencing are continually under development. Assays using the polymerase chain reaction (PCR) have become the mainstay for the detection of filoviruses in outbreak settings. In many cases, real-time reverse transcriptase-PCR allows for the detection of filoviruses to be carried out with minimal manipulation and equipment and can provide results in less than 2 h. In cases of novel, highly diverse filoviruses, random-primed pyrosequencing approaches have proved useful. Ideally, diagnostic tests would allow for diagnosis of filovirus infection as early as possible after infection, either before symptoms begin, in the event of a known exposure or epidemiologic outbreak, or post-symptomatically. If tests could provide an early definitive diagnosis, then this information may be used to inform the choice of possible therapeutics. Several exciting new candidate therapeutics have been described recently; molecules that have therapeutic activity when administered to animal models of infection several days post-exposure, once signs of disease have begun. The latest data for candidate nucleoside analogs, small interfering RNA (siRNA) molecules, phosphorodiamidate (PMO) molecules, as well as antibody and blood-product therapeutics and therapeutic vaccines are discussed. For filovirus researchers and government agencies interested in making treatments available for a nation's defense as well as its general public, having the right diagnostic tools to identify filovirus infections, as well as a panel of available therapeutics for treatment when needed, is a high priority. Additional research in both areas is required for ultimate success, but significant progress is being made to reach these goals.

A single phosphorodiamidate morpholino oligomer targeting VP24 protects rhesus monkeys against lethal Ebola virus infection.

UNLABELLED: Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMOplus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMOplus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures. IMPORTANCE: Several West African countries are currently being ravaged by an outbreak of Ebola virus (EBOV) that has become a major epidemic affecting not only these African countries but also Europe and the United States. A better understanding of the mechanism of virulence of EBOV is important for the development of effective treatments, as no licensed treatments or vaccines for EBOV disease are currently available. This study of phosphorodiamidate morpholino oligomers (PMOs) targeting the mRNAs of two different EBOV proteins, alone and in combination, demonstrated that targeting a single protein was effective at conferring a significant survival benefit in an EBOV lethal primate model. Future development of PMOs with efficacy against EBOV will be simplified if only one PMO is required instead of a combination, particularly in terms of regulatory approval.