Sudan Virus Research Articles

Immune memory to Sudan virus: comparison between two separate disease outbreaks.

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda—Gulu 2000–2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1–649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1–649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.

Identification of Invariant Peptide Domains within Ebola Virus Glycoprotein GP1, 2

Six invariant oligopeptide sequences of length 12 amino acids, or greater, were identified in GP1,2 of ZEBOV Zaire and SEBOV Sudan Ebola viruses. Five of these invariant peptides had positive scores for predicted B-cell epitope scores. In contrast, the amino acid positions of the remaining invariant peptide all displayed negative predicted epitope scores. Invariant regions of Ebola GP1,2 oligopeptides may reflect structural, functional and immunological constraints on the virus, and thus, may be useful as immunological and pharmacological targets.

Investigating the zoonotic origin of the West African Ebola epidemic.

The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2-year-old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free-tailed bats (Mops condylurus). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology.

Ebola Virus Disease: virology, pathogenesis, therapy, and vaccines

2013年12月始， 埃博拉病毒病在几内亚、利比里亚、塞拉利昂和尼日利亚4国暴发. 其病原体--埃博拉病毒（Ebolavirus）于1976年被首次分离， 为单股负链、不分节段、有囊膜的RNA病毒， 属丝状病毒科， 分5种， 包括扎伊尔型、苏丹型、本迪布焦型、塔伊森林型和莱斯顿型. 其基因顺序为3'端-NP-VP35-VP40-GP-VP30-VP24-L-5'端. 埃博拉病毒属于A类病原， 致病能力极强， 操作活病毒需在生物安全4级实验室进行. 其主要靶细胞是血管内皮细胞、肝细胞、巨噬细胞和树突状细胞等， 通过抑制天然和获得性免疫应答反应， 增加血管通透性， 引起肝脏等多脏器损伤， 引发发热、出血、多器官功能衰竭和休克等症状. 目前尚无批准上市的特异治疗药物和疫苗， 处于研发或临床试验阶段的治疗药物包括： （ⅰ） 抗流感病毒药物T-705可能阻碍埃博拉病毒在细胞内增殖， 从而遏制感染； （ⅱ） ZMapp包含3种人源化的单克隆抗体， 属优化的鸡尾酒疗法； （ⅲ） 基于RNA干扰的基因治疗药物TKM-Ebola； （ⅳ） 凝血调节因子、炎性介质制剂， 如源于丝虫的抗凝蛋白rNAPc2等. 处于研发阶段的疫苗有： （ⅰ） 复制缺陷型疫苗， 安全性好， 但诱导免疫的时间较长； （ⅱ） 减毒复制型疫苗， 效果好， 但存在安全隐患. 开展深入的病原学、免疫病理和致病机制研究， 将有利于治疗药物和疫苗的研发. 同时， 应推进有希望的治疗方案和疫苗进入人体临床试验阶段.

Biomarker Correlates of Survival in Pediatric Patients with Ebola Virus Disease

Outbreaks of Ebola virus disease (EVD) occur sporadically in Africa and are associated with high case-fatality rates. Historically, children have been less affected than adults. The 2000-2001 Sudan virus-associated EVD outbreak in the Gulu district of Uganda resulted in 55 pediatric and 161 adult laboratory-confirmed cases. We used a series of multiplex assays to measure the concentrations of 55 serum analytes in specimens from patients from that outbreak to identify biomarkers specific to pediatric disease. Pediatric patients who survived had higher levels of the chemokine regulated on activation, normal T-cell expressed and secreted marker and lower levels of plasminogen activator inhibitor 1, soluble intracellular adhesion molecule, and soluble vascular cell adhesion molecule than did pediatric patients who died. Adult patients had similar levels of these analytes regardless of outcome. Our findings suggest that children with EVD may benefit from different treatment regimens than those for adults.

Development and evaluation of a panel of filovirus sequence capture probes for pathogen detection by next-generation sequencing.

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.

Pandora of Ebola virus: are we ready?

On 23 March 2014, the ministry of health of Guineanotified WHO of a rapidly spreading outbreak of Ebolavirus disease (EVD) in south-eastern forested areas, and4 days later, the disease was confirmed to be transmitted toConakry, the Capital of Guinea. The disease also rapidlyspread to Liberia, Nigeria and Sierra Leone. As of 27 July2014, the cumulative number of confirmed (909), probable(276) and suspect (138) EVD cases in the above fourcountries reached 1,323, including 729 deaths (with thedeath rate of 55.1 %) (http://www.who.int/csr/don/2014_07_31_ebola/en/). This is the largest outbreak of EVD untilnow and the first time the disease detected in West Africa.EVD, previously known as Ebola haemorrhagic fever, isa severe, often fatal illness, with very high case fatality rateof up to 90 %. The previous EVD outbreaks in Uganda(epidemic from October of 2000 to February of 2001) andin Gabon and the Republic of the Congo (outbreak fromDecember of 2001 to March of 2002) were also persistedfor several months. The outbreak of EVD in West Africaseemed out of control although WHO has coordinated withdifferent organizations and countries for containing thisdisease [1]. For the last four months, the EVD epidemic hasspread to the nearby countries and new cases and newdeaths were reported almost every day. Until 6 August2014, there were 14 outbreaks of EVD and three singlecase reports in the world, resulting in 4,178 cases ofinfection and 2,564 deaths, with death rate from 41 %–88 %(excluding the single case report, Fig. 1).Ebola was first reported in 1976 when there were out-breaks in Sudan and Democratic Republic of Congo [2, 3].The genus Ebolavirus belongs to the Filoviridae family(filovirus), which also includes genus Marburgvirus andgenus Cuevavirus. There are five distinct species in thegenus Ebolavirus, including Bundibugyo ebolavirus; Zaireebolavirus; Sudan ebolavirus; Reston ebolavirus; and Tai¨Forest ebolavirus. The first three species have been asso-ciated with large EVD outbreaks in Africa, whereas thelatter two have not. Samples taken from patients of thisoutbreak have been confirmed to be caused by a strain ofebolavirus closely related (98 %) to the Zaire one. Theebolavirus should be operated under biosafety level IVlaboratory. Even in this high-level containment laboratory,there were still accidentally laboratory-acquired infectionsreported [4, 5].Fruit bats are believed to be the natural hosts of Ebolavirus [6]. The wild animals (monkeys or apes) could becontracted the virus from contacting with bat saliva orfaeces. Humans could be transmitted from contacting withinfected bats or wild animals. The EVD can be transmittedfrom person to person by direct contact. More and moreevidence showed that pigs might be the host of ebolavirus[7, 8]. From the distribution of fruit bats (http://www.who.int/csr/disease/ebola/en/#), the potential riskregion of EVD not only is limited to Africa but alsoincludes wide range of America, southern Asia and part ofAustralia.Since there is no effective antiviral drugs and specificvaccine for treating and preventing EVD, we all need toprepare for early recognition of EVD and special pre-ventive countermeasures should be taken for the suspectedpatients. Rapid and specific screening assays should bedeveloped for early diagnosis of EVD; however, the clin-ical doctors should pay close attention to the suspectedpatients, especially for those with travel history in theepidemic regions. Hospitals need to prepare to protect

Synthetic antibodies with a human framework that protect mice from lethal Sudan ebolavirus challenge.

The ebolaviruses cause severe and rapidly progressing hemorrhagic fever. There are five ebolavirus species; although much is known about Zaire ebolavirus (EBOV) and its neutralization by antibodies, little is known about Sudan ebolavirus (SUDV), which is emerging with increasing frequency. Here we describe monoclonal antibodies containing a human framework that potently inhibit infection by SUDV and protect mice from lethal challenge. The murine antibody 16F6, which binds the SUDV envelope glycoprotein (GP), served as the starting point for design. Sequence and structural alignment revealed similarities between 16F6 and YADS1, a synthetic antibody with a humanized scaffold. A focused phage library was constructed and screened to impart 16F6-like recognition properties onto the YADS1 scaffold. A panel of 17 antibodies were characterized and found to have a range of neutralization potentials against a pseudotype virus infection model. Neutralization correlated with GP binding as determined by ELISA. Two of these clones, E10 and F4, potently inhibited authentic SUDV and conferred protection and memory immunity in mice from lethal SUDV challenge. E10 and F4 were further shown to bind to the same epitope on GP as 16F6 with comparable affinities. These antibodies represent strong immunotherapeutic candidates for treatment of SUDV infection.

Molecular characterization of a naturally occurring intraspecific recombinant begomovirus with close relatives widespread in southern Arabia.

BackgroundTomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed.FindingsThe complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants.ConclusionFive new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.

T and B cell memory responses in 2012 filovirus outbreak survivors (VIR7P.1060)

Abstract From 2011 to the present, there have been four confirmed filovirus outbreaks with a fifth case involving a single patient. Four outbreaks/cases were attributed to the Ebolavirus genus, with Marburgvirus responsible for one. With the high lethality rates seen in these outbreaks (generally ~40-90%), and the potentially dramatic disease sequelae, filovirus outbreaks can devastate local communities and present great health risks for affected regions. These issues obviate the need for effective vaccines and therapeutics to limit and manage future outbreaks. In 2012 alone, three outbreaks occurred in Uganda - two of Sudan virus and one Marburg virus. We collected PBMC and serum samples from survivors from each outbreak to evaluate the presence of immune memory and its composition. Serum antibodies displayed broad specificity to Marburg virus and Sudan virus glycoprotein in addition to other proteins. Virtually all serum samples displayed neutralizing activity. PBMCs stimulated with viral and recombinant antigen were evaluated by flow cytometry for functional parameters as well as level of antigen-responsive T cells. Cytokines in the supernatants of these cultures were determined by multiplex ELISA. Cytokine responses were largely pro-inflammatory produced predominantly by CD4 T cells. Establishing immune memory parameters and the nature thereof gives us targets for vaccine development and opens avenues for development of immune-based therapeutics.

Vesicular Stomatitis Virus-Based Vaccines Protect Nonhuman Primates against Bundibugyo ebolavirus

Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29–36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.

A Distinct Strain of Tomato leaf curl Sudan virus Causes Tomato Leaf Curl Disease in Oman.

Tomato leaf curl disease (ToLCD) is a significant constraint for tomato production in the Sultanate of Oman. The disease in the north of the country has previously been shown to be caused by the monopartite begomoviruses (family Geminiviridae) Tomato yellow leaf curl virus and Tomato leaf curl Oman virus. Many tomato plants infected with these two viruses were also found to harbor a symptom enhancing betasatellite. Here an analysis of a virus isolated from tomato exhibiting ToLCD symptoms originating from south and central Oman is reported. Three clones of a monopartite begomovirus were obtained. One of the clones was shown to be infectious to tomato and Nicotiana benthamiana and to induce symptoms typical of ToLCD. Analysis of the cloned sequences show them to correspond to isolates of Tomato leaf curl Sudan virus (ToLCSDV), a virus that occurs in Sudan and Yemen. However, the sequences showed less than 93% nucleotide sequence identity to previously characterized ToLCSDV isolates, indicating that the viruses represent a distinct strain of the species, for which we propose the name "Oman" strain (ToLCSDV-OM). Closer analysis of the sequences showed them to differ from their closest relative, the "Tobacco" strain of ToLCSDV originating from Yemen, in three regions of the genome. This suggests that the divergence of the "Oman" and "Tobacco" strains has occurred due to recombination. Surprisingly, ToLCSDV-OM was not found to be associated with a betasatellite, even though the isolates of the other ToLCSDV strains have been shown to be. The significance of these findings and the possible reasons for the distinct geographic distributions of the tomato-infecting begomoviruses within Oman are discussed.

Cotton leaf curl Gezira alphasatellite associated with tomato leaf curl Sudan virus approaches the expected upper size limit in the evolution of alphasatellites

Begomoviruses (family Geminiviridae) cause severe damage to tomato crops worldwide. Among them, tomato leaf curl disease (ToLCD)-associated begomoviruses are a major concern for tomato production in Sudan. Here, we report the detection of unexpectedly large cotton leaf curl Gezira alphasatellite molecules (up to 1467 nt) associated with an isolate of a novel strain of tomato leaf curl Sudan virus (ToLCSDV) in tomato plants affected by ToLCD. A recombinant nature is suggested for this ToLCSDV isolate.

Induction of Broad Cytotoxic T Cells by Protective DNA Vaccination Against Marburg and Ebola

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.

Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012

In 2012, an unprecedented number of four distinct, partially overlapping filovirus-associated viral hemorrhagic fever outbreaks were detected in equatorial Africa. Analysis of complete virus genome sequences confirmed the reemergence of Sudan virus and Marburg virus in Uganda, and the first emergence of Bundibugyo virus in the Democratic Republic of the Congo.

Conformational properties of peptides corresponding to the ebolavirus GP2 membrane-proximal external region in the presence of micelle-forming surfactants and lipids.

Ebola virus and Sudan virus are members of the family Filoviridae of nonsegmented negative-strand RNA viruses ("filoviruses") that cause severe hemorrhagic fever with fatality rates as high as 90%. Infection by filoviruses requires membrane fusion between the host and the virus; this process is facilitated by the two subunits of the envelope glycoprotein, GP1 (the surface subunit) and GP2 (the transmembrane subunit). The membrane-proximal external region (MPER) is a Trp-rich segment that immediately precedes the transmembrane domain of GP2. In the analogous glycoprotein for HIV-1 (gp41), the MPER is critical for membrane fusion and is the target of several neutralizing antibodies. However, the role of the MPER in filovirus GP2 and its importance in membrane fusion have not been established. Here, we characterize the conformational properties of peptides representing the GP MPER segments of Ebola virus and Sudan virus in the presence of micelle-forming surfactants and lipids, at pH 7 and 4.6. Circular dichroism spectroscopy and tryptophan fluorescence indicate that the GP2 MPER peptides bind to micelles of sodium dodecyl sulfate and dodecylphosphocholine (DPC). Nuclear magnetic resonance spectroscopy of the Sudan virus MPER peptide revealed that residues 644-651 interact directly with DPC, and that this interaction enhances the helical conformation of the peptide. The Sudan virus MPER peptide was found to moderately inhibit cell entry by a GP-pseudotyped vesicular stomatitis virus but did not induce leakage of a fluorescent molecule from a large unilammellar vesicle comprised of 1-palmitoyl-2-oleoylphosphatidylcholine or cause hemolysis. Taken together, this analysis suggests the filovirus GP2 MPER binds and inserts shallowly into lipid membranes.

Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA) formulation.

BackgroundAntigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism.Methods and FindingsIn parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections.ConclusionsThe C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus.

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

Cathepsin B & L Are Not Required for Ebola Virus Replication

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB−/− and catL−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

A Characterization of Aerosolized Sudan Virus Infection in African Green Monkeys, Cynomolgus Macaques, and Rhesus Macaques

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and “Cuevavirus”. Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4–5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP.