A review on the antagonist Ebola: A prophylactic approach

Ebola virus (EBOV), a member of Filoviridae virus family under the genus Ebolavirus, has emerged as a dangerous and potential threat to human health globally. It causes a severe and deadly hemorrhagic fever in humans and other mammals, called Ebola Virus Disease (EVD). In recent outbreaks of EVD, there has been loss of large numbers of individual’s life. Therefore, EBOV has attracted researchers and increased interests in developing new models for virus evolution, and therapies. The EBOV interacts with the immune system of the host which led to understand how the virus functions and effects immune system behaviour. This article presents an exhaustive review on Ebola research which includes EVD illness, symptoms, transmission patterns, patho-physiology conditions, development of antiviral agents and vaccines, resilient health system, dynamics and mathematical model of EBOV, challenges and prospects for future studies.

Introduction
Ebola virus (EBOV) has emerged as a potential threat to human health worldwide, which cause a critical and serious ailment, often deadly if not cured properly. It is a member of "Filoviridae" virus family, under the genus "Ebolavirus", having five species: Zaire Ebola, Tai Forest Ebola, Sudan Ebola, Bundibugyo Ebola, and Reston Ebola Virus [1]. Out of these species, former four cause illness in humans, whereas the Reston Ebola Virus causes illness in primates. Ebola virus causes an illness called as hemorrhagic fever [2], especially in humans and other mammals, named as "Ebola Virus Disease" (EVD), and therefore EVD is also known as "Ebola Hemorrhagic Fever" (EHF). Ebola viruses are native to a number of African countries. The disease was firstly recognized in 1976 in two concurrent outbreaks: one of them in Nzara, an state in South Sudan, and another one in village Yambuku, previously known as Democratic Republic of Congo, adjacent to 'Ebola River', and hence the name Ebola Virus Disease [3].

EBOV and the immune system
EBOV is an enveloped virus having a 19 kb single-stranded, negative-sense RNA genome which encodes 7 proteins [4]. The core of the virus is composed of the RNA genome, called nucleoprotein (NP), that covers the genomic RNA and nucleocapsid viral protein 30 (VP30) which is covered by a lipid envelope having projected surface composed of a glycoprotein (GP). The surface glycoprotein is a multimer having roles in cell attachment, fusion and cell entry, helps in immune evasion and pathogenesis of disease [5]. The diagrammatic representation of Ebola genome is shown in Fig. 1.
Zaire Ebola Virus triggers the immune response and then punctures the vascular system. When the virus enters the body, it infects various types of immune cells by initially targeting monocytes and macrophages, and cause the release of proinflammatory cytokines and chemokines [98]. In the previous studies it was shown that host Toll-like receptor 4 (TLR4) is a sensor for EBOV glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. EBOV GP interacts with TLR4, and on VLPs it was able to stimulate expression of NF-κB in a TLR4-dependent manner. It was also found that budding of EBOV VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. These findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP and play an important role in the immune-pathogenesis.
The immune system forms a complex network of cells communicating to each other with the help of soluble mediators such as cytokines. Immune system has both innate and adaptive immunities to protect the body against pathogens. Nucleotides from RNA viruses are recognized by retinoic acid inducible gene I (RIG-I)-like helicases (RLHs) and Toll-like receptors (TLRs). It triggers signaling cascades that induce anti-viral mediators such as type I interferons (IFNs) and proinflammatory cytokines.
In many viral infections, the early action of cytokines produced by infected cells and dendritic cells is sufficient to eliminate the pathogen [6]. In some cases, innate defenses are not able to handle viral infection, and second-line of defenses is mobilized to ensure host survival. The adaptive defense consists of antibodies and lymphocytes, often called the humoral response and the cell mediated responses which are essential for destruction of viruses [6]. Innate and adaptive responses usually work together to eliminate the viruses, but in some cases both these immune responses are not able to eradicate the viruses which led to diseases. Filoviridae family of viruses is one of the examples which are not destroyed by the immune system. Many studies shown that the induction of an innate immune response lead to infection or stimulation of macrophages/monocytes and DCs with Ebola virus or VLPs, respectively. For example, incubation of Ebola virus VP40 + GP VLPs with DCs led to the induction of interleukin-6 (IL-6), IL-8, NF-κB and ERK1/2. Ebola virus VP40 + GP VLPs, but not VP40 VLPs, induced cytokine and SOCS1 expression in a TLR4/ MD2 dependent manner both in a human monocytic cell line (THP-1 cells) and in 293T cells expressing a functional TLR4/MD2 receptor. Stimulation of TLR4 by Ebola virus envelope GP results in an innate host response, induction of SOCS1 protein, and potential enhancement of virus egress. The detailed coverage of various genes involved in immune response can be found in [5,6,7,46].
There were several EVD outbreaks in the past years, but the most severe and the largest was the recent widespread outbreak in West Africa (2013-2016), having 28,616 cases and 11,310 deaths, and estimated case fatality rate of 71% [8,9]. Fig. 2 presents country and yearwise outbreaks, number of cases registered, and number of deaths..

Clinical features and complications
Signs and symptoms of EVD start between 2-21 days after contacting the virus, and the most common symptoms are fever, sore throat, muscular pain and headache [10] (Fig. 3). With high concentration of virus, vomiting, diarrhoea, and rashes are usually occurred which decrease the function of liver and kidneys. It also causes bleeding both internally and externally which lead to high risk of death and killing 90% of infected people due to low blood pressure from body fluids [11]. The severity of symptoms of EVD compared with ZikV [12], Dengue, Chikungunya and West Nile are presented in Table 1.
Ebola virus is responsible for haemorrhagic fever leading to various complications such as malaise, fever, vascular permeability, decreased plasma volume, coagulation abnormalities, and varying degrees of hemorrhage [13]. It is also notified that the incubation period of filoviruses is 2-21days after infection, with identifying symptoms such as chills, fever, myalgia and malaise, which is followed by lethargy, nausea, vomiting, abdominal pain, anorexia, diarrhea, coughing, headache, hypotension, maculopapular rash and mucosal bleeding, in the gastrointestinal and genitourinary tracts. With the pertaining of these diseases, specially as a result of hypotensive shock and multiorgan failure (including hepatic damage and renal failure), death usually occurs in 6-16 days after the onset of symptoms [14].
In a statistical study of EVD cases [15], the amount of survived and died individuals are determined by various parameters such as age, sex and symptoms as shown as charts in Fig. 4. It can be inferred from Fig. 4 that mortality rate of EVD patients are higher in males than females. Similarly, the bleeding events from faeces show a high mortality rate during admission as well as hospitalization.   Several people have survived from EVD during the previous major outbreaks in Sierra leone (2014-16). However, many complications of EVD and its factors persist and responsible for death [16].Various signs and symptoms of EBOV infection has been associated with many complications [16,17], given in Table 2.
The most common and frequent complications were Neurological problems (brain as well as spinal cord problems), Ocular complications (eye difficulties), and Musculoskeletal problems (including body's muscles problems) [18].

EBOV transmissions
EVD is a zoonotic disease which involves animals and humans [19]. However, there is no evidence of the natural reservoir host. The scientists investigated that the fruit bats are the carrier hosts of the EBOV and usually contain the virus particles. The viruses which were carried by bats are transmitted to animals, such as monkeys, apes, forest's antelopes, and also to humans, who came in contact with the bodily fluids like blood and secretions of such infected organisms. These transmissions are possible between animal-to-animal, animal-tohuman, and human-to-human (Fig. 5).
In addition to infected skin, EVD also transmits through bodily fluids such as blood and secretions of infected people, and with some kind of objects such as beds and clothes spoiled with such type of fluids [19]. The EVD patients also cause illness to many health-care workers during care and treatment. Transmission of EBOV has also been reported when people attend traditional funeral practices which involve direct contact with the body of the deceased people (Fig. 5).

EVD epidemics and persistence of virus after recovery
After the transmission of Ebola virus to different part of the West Africa such as Guinea, Liberia, and Sierra Leone, epidemic of Ebola virus disease had resulted in a total of 28,646 cases and 11,323 deaths. In addition, Ebola cases were also exported to several other African and European countries including United States, with further transmission to healthcare workers across several countries [13]. The large number of Ebola survivors has the frequency of persistent symptoms and the possibility of virus persistence in sanctuary sites, leading to delayed transmission. EBOV may also persist in immune-privileged sites and semen of survivors which lead to re-ignition of EVD outbreak. As EBOV RNA can remain detectable in immune-privileged tissues for prolonged periods of time after clearance from the blood, suggesting that it may persists during convalescence and thereafter [97]. Eliminating persistent EBOV is important to ensure full recovery of survivors and decrease the risk of outbreak re-ignition caused by EBOV spread from healthy survivors to new contact [20].
Due to persistence of EVD in West Africa, most of the survivors have many residues of infection of disease and complications, such as arthritis and vision-threatening uveitis [97]. Many of the symptoms such as fatigue, sleep disturbance, blurred vision, retro-orbital pain, hearing loss, difficulty swallowing, and muscle weakness were reported after 21 months of follow-up. This lead to delayed clearance of viruses in selected and other body compartments, semen of the men, pregnant women, fetuses, products of conception, and breast milk [21].

Dynamics of the mechanism of EBOV
There is a very interesting relationship between the ecosystem and the human population, where ecosystem is like a natural home for humans. Wildlife plays an important source of infectious diseases for the human race. There are myriads of serious, harmful zoonotic infections that have been caused by bats (vector), for example, EBOV, Marburg, SARS-corona virus, Nipah, Hendra, multiple types of rabies etc. There is an intrinsic relationship between the ecosystem health and  the human health which, unfortunately, has not been understood well [22,23]. Our ecosystem is not just composed of the goody-goody beings, but also some of the most threatening pathogens, such as RNA viruses. These viruses are very harmful as they have the potential to adapt to new hosts and environments [24]. Henceforth, we have a challenge to protect the wildlife and their habitats, by that safeguarding vital ecosystem structures and functions having local as well as broader consequences for human wellbeing [25].There are high public health risks, disease threats which are because of RNA viruses associated with fruit bats. The spill-over of infections occurs from bats to humans, but the compendium of dynamics involved depends on the extent of spill-over that has been recognized and understood and revolves around three important factors: Biological Factors, Environmental Factors, and Social Factors.
Zoonosis related health viruses also focus less studied social and environmental queries, like why are bats the only vectors for EBOV? How do people interact with bats, both directly and indirectly? What are the environmental factors that force viral pathogen dynamics and spill-over between many species of bats? To what extent bats cause infectious diseases in human being? How do the above mentioned factors influenced in the spread of the diseases? Does the bats ancestry, its life history, and feeding habits play a role in deciding whether it is capable of becoming a vector or not? Are bats the only ones which act as a cave for many deadly viruses, or are there more vectors? To answer some of these questions, biological factors have been studied in detail, but there is a need to study the social and environmental factors. For this, it is mandatory to take up a holistic approach of study [25].

Need of the hour: the holistic approach
An integrated study of the three factors: Biological, Social, Environmental is mandatory to understand and explain the actual mechanism of zoonotic emergence. Fig. 6 depicts and describes the holistic approach to understand zoonotic disease like EVD. We need to understand the virus evolution, transmission and its spill-over to humans using mathematical and ecological models. The dynamics of individual spill-over episodes are based around the notion of the pyramid (the triangle that has been shown in Fig. 7) of pathogen emergence. It is suggested that humans are confronted by animal derived pathogens, however, only a small fraction of people (having weak immunity) get affected. Spill-over dynamics are liable to a range of local impact and practice, both social and environmental influences on viral pathogen dynamics in the manner that interaction with susceptible sympatric species [25]. These models give us a skeletal framework for studying the dynamics of infections in bat reservoir populations [26].

Role of systems biology in elucidating the framework
Using advanced computational and analytical techniques, we can capture and develop a framework that adds the dynamic relationship between the virus (EBOV), bats, animals (candidate hosts) and humans  (target) in a local system of three factors explained in Fig. 7. Systems Biology can be used for modeling the events, and deriving the answers to all those questions which are still unanswered. Systematic Modeling and Interaction Modeling can be used extensively to model biological phenomenon. With the help of computational and analytics approaches, we can unlock many new theories related to EVD transmission, and mechanism of EBOV invasions. We can even develop better vaccines and drugs for the diseases [27]. Infectious diseases are growing rapidly as the pathogens are evolving themselves against our therapeutics. There has been an urgent need for improved remedies against pathogens. Systems Biology based studies have proved to be icing on the cake! Systems Biology based analysis have been worked out recently in order to capture how the host genes are associated with the viral replication that tend to occupy space in the human protein interactome [28,29]. A comprehensive systems-level analysis of the virus-host interactome is very important for understanding the roles of host factors Many antiviral drug discovery approaches have produced appreciable achievements but there are no medications available in order to fight it. Systems biology based antiviral drug studies have tremendously helped in the identification of new antiviral candidates. Cheng et al. [31] have proposed an integrated approach to determine new antiviral candidates for the existing drugs by adding drug-gene patterns into the virus-host interactome. Using systems biology analysis they have determined three molecule drugs namely, Ajmaline, Piroxicam and Azlocillin, as the new drug candidates for anti-Ebola virus treatment. Along with this, they have also proposed the mechanism showing how these drugs can block the Ebola virus and henceforth, inhibit the infection.
In addition, software tools are also developed to identify EVD patients, along with the risk of EVD suffering in the future. Ebinformatics Software [32], known to be Ebola Fuzzy Informatics system, is developed in order to diagnose, prescribe appropriate medications to EVD patients. Based on the user evaluation result, this software has proven to be a valuable addition to the operational and research enterprise to fight against Ebola [32]. This multifaceted yet single platform has been achieved by gathering information such as factors that cause Ebola-like signs and symptoms, and a system was developed based on facts for the prognosis and diagnosis of the disease. This system relates the conditions and answer whether one is suffering from EVD or not.

Need to correlate the three factors
These are the biological, social, and environmental factors that are responsible for the disease spread. Such factors, as mentioned earlier, are not understood well for most of the zoonotic diseases. Thus, there is a need for some sort of empirical modeling techniques which can elucidate and derive a correlation of disease spill-over and their forces at various levels [27,33]. Spatially categorical ecosystem data, including wildlife densities, livestock densities, human population densities, climate and socio-economic variables are mostly accessible for such analyses [27,33].
In a nutshell, there is an urgent need for a framework for the holistic study of diseases caused by bats as the vector. Interdisciplinary studies play a vital role in mining the zoonotic diseases. The collaboration between Life Sciences, Mathematics, Systems Biology, Computational and Analytical approaches is a must for studying the disease dynamics. This will also help us to understand how to cope up with such apex diseases, and preventing them from spreading further.

Clinical strategies and challenges in the treatment
The most recent and the biggest epidemic implicating more than 28,000 cases in West Africa was triggered by a variant strain of EBOV with an approximate global death of ∼40% [34]. We lacked the certified therapeutics to treat EVD during 2014-15 outbreaks which demolished West African countries [35].
As diagnostic techniques for EBOV, Enzyme-linked immune-sorbent assay (ELISA) and real-time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) are the mostly used. Antigen or antibody (IgM/IgG) is ascertained by ELISA and rRT-PCR through which presence of Ebola virus-specific genes are identified after virus isolation. However, antibody-capture ELISA method is restricted to be applied during early diagnosis due to non-appearance of antibodies within 1-2 week(s) of illness [36]. The developmental stages of therapeutics and drugs against EBOV have been very slow as most of the procedures related to the research and development has always been tricky and a big challenge, plus, it requires bio-safety level-4 laboratories.
Though a number of vaccines and anti-viral drugs have been developed against EBOV which provided satisfying results only for the first phase of clinical trials, but phase II, III and IV studies didn't pass the trial lately. This failure highlights the fact that we still need better equipment, resources and technology to develop an optimized treatment for preventing and curing EVD. International organizations are establishing treatment centers which must be prepared to enlist patients for clinical trials, and have appropriate structures for the management of ethical and medico-legal requirements [37,38].
Other challenges to conduct clinical trials are the need to train personnel to enter the "red zone" of Ebola Treatment Centres, thus, avoiding from risk exposure to Ebola, seeking consent from serious EVD   patients for complex studies, and managing and expediting ethical review process between various governmental and non-governmental healthcare/research organizations [39]. The following section mentions known therapeutics which comprises of vaccines, small molecule based therapies, antibody based therapies, and also showcases how far we have reached in developing latest vaccines and drugs to eliminate EBOV [40].

Symptomatic treatment against Ebola
Symptomatic treatment refers to the immediate and urgent treatment that is given when a person gets infected. This treatment is completely dependent on clinical requirements, viz. oral/intravenous therapy, analgesics, antipyretics, anti-diarrheal medications, antipsychotic drugs etc.., as per the patient's signs and symptoms [41,90]. Table 3 summarizes detailed information about the medications that are used for the symptomatic treatment along with the side effects that may arise.

Therapeutics involved in treatment
Vaccinations are known to play a pivot role in reducing risks of many diseases, including EVD. Since the rise of biotechnology, pharmaceuticals have also used the antibodies (IgG/IgM) and other small molecules (lipids) to reduce the risk of Ebola, summarized details is given in Table 4.Vaccination alone is no "cure-all remedy". We need to have a better management system which abstains itself from all the bigotry and favoritism.
Drug discovery and development is a very expensive process in both the terms time and money. Once such medications are ready to be circulated, these medications are not easily available to the low economic countries. Therefore, it is an utmost responsibility of the pharmaceutical industries and the governments to join their hands and to make such medications available at cheap costs. After all, a human being is a resource, and a life saved is more important than to make heaps of stocks of money. The current drugs that are being used to treat Ebola affected patients are so costly, for example: Favipiravir (T-705) 25mg costs around $280, whereas, the same medicine but of a higher dosage, about 100mg costs around $780. Such therapeutic treatments and medications are very costly, which actually can be one of the reasons why we still lag behind in completely eliminating Ebola roots from the world. Epidemiologic practices, like toss-back analyses to establish and lazaretto people debunked to Ebola, are momentous to curb the spill-over. Such control practices are desired to train people on the ground in even the most lonesome locale [87]. Given that nosocomial transmission has contributed heavily to past Ebola epidemics [42]. It is also indispensible to coalesce vaccination with nosocomial contact precautions and quarantining [43,44].
Several problems were faced by the scientists while evaluating the drugs mentioned in Table 4. Before testing any drug, patients need to be isolated and there should be no contact between the patients and the rest of the people, which can cause further contamination [98]. The blood samples are taken and rapidly sent to the pathological laboratories for testing. In the fresh phase of the disease, fluid replacement therapy hysterically escalates the probability of endurability. Ribavirin, the only acknowledged antiviral viz. competent against certain VHF pathogens such as Lassa fever, is not active against Ebola viruses, which is unfortunate as it has already passed the 3rd phase of clinical trials. ZMapp, on the other hand, is a combination of monoclonal antibodies used to treat Ebola patients. Its role in treating EVD is still not certified as enough data proving it to be efficient is yet not published. It works well when tested on non-primates as it was capable enough to revert back the EVD. Favipiravir is the most commonly used and tested drug on humans. When tested on mice, it prevented its death, but its activity in non-human models still need to be validated. BCX 4430 is another drug extensively studied and worked on, and has proved to be effective against Marburg Viruses in non-human primate models and Ebola model of mice. Ebola outbreak of Kikwit in 1995 was treated with patients being treated to Convalescent Plasma treatment, which at that time proved to be better as fewer people died and many survived. These days, with latest techniques and technology, this treatment is not being used much [45].
Other drugs such as TKM, AVI6002, Amiodarone, Lamivudine, Statins, Clomiphene, U18666-A, and Novavax are still under developed (Phase-I Morphine, Fentanil (fentanyl) Morphine is an opiate alkaloid extracted from the Poppy plant (Papaver somniferum). It binds and activates specific opiate receptors that are involved in controlling most of the functions of brain. Fentanil is a synthetic, lipophilic phenyl piperidine opoid agonist anesthetic and analgesic property.
(i) Morphine, if taken in excess may cause drowsiness and dizziness. (ii) Fentanil if swallowed, may be fatal, cause breathing difficulties, skin allergies, damages fertility and harm to breast fed children.
Diarrhoea Imodium (Loperamide) Loperamide is a synthetic agent chemically related to opiates with anti-diarrheal properties.
Toxic if swallowed.
Vomiting / Nausea Metaclopramide, Ondansetron; some physicians may also recommend NG tube (Nasogastric Intubation) Metaclopramide is a substituted benzamide and a derivative of para-aminobenzoic acid (PABA), with gastroprokinetic and anti-emetic effects. Ondansetron is a competitive serotonin type 3 receptor antagonist viz. used to treat nausea and vomiting.
(i) Metaclopramide may have reproductive toxicity.
(ii) Ondansetron generally causes headaches, constipation and dizziness, and skin irritations.

Psychotic Episodes Diazepam, Haloperidol
Diazepam is a benzodiazepine derivative with sedative, hypnotic and anticonvulsant properties. Haloperidol is a phenyl piperdinyl-butyrophenone that is used to treat schizophrenia and other psychoses.
• Diazepam is suspected to cause cancer.
• Haloperidol causes skin, eyes and respiratory irritations, and reproductive toxicity.

Hiccups
Chlorpromazine may also be treated by using NG tube.
It is basically an anti-psychotic drug but has many other properties, it is used as an anti-emetic drug and it also used in the treatment of intractable hiccups.
Acute toxicity and causes skin problems.   and Phase-II of clinical trials). These drugs have been proven to be effective against mice but there is no trace of their efficacy in humans. Both developed and developing therapeutics are under clinical trials and have some Achilles heels, but most of them have passed phase II trials. It means that they are capable enough of treating Ebola patients, but at the same time it needs to be suitable enough and efficient enough to work on the human body. These drugs pass the test on animal, nonprimates human models, but usually fail to respond testing on human because of its complex mechanism. Furthermore, EBOV attacks the innate and the adaptive immunity, thereby creating chaos which restricts these drugs in the human body.

Mode of vaccine delivery
Vaccines can be delivered using two main routes: Cytomegalovirus (CMV) based vaccination, and Intranasal Route. The CMV-based vaccination has been studied in rodents [46,47]. A single dosage of CMV expressing a CD8T cell epitope from nucleoprotein of EBOV persuaded durable CD8 + T cell immunity up to 33 weeks. No EVD was detected in vaccinated mice. These mice did, nevertheless, confirm loss of weight, implying protection [39]. The intranasal route was suggested as an effective vaccine delivery route [48]. This approach has the potential to escalate compliance, especially in populations dubious of Western therapeutics and medications [39].

Candidate vaccines
There are several vaccines which are developed, but Live Attenuated Vaccines are aforethought and examined as an aboriginal, a competitor, full of potency, and asylum were tested using nonhuman anthropoids as primary animal figurines. Despite, the results did not ascertain any cogent vaccine candidates even in commensuration of animal model [92]. Nevertheless, Ebola vaccine design and development studies have been going on for the last several decades, but unfortunately, the pace was not so rapid or rewarding. As a result, the development of vaccines related to EBOV has several limitations related to safety and personal expertise [36]. Some approaches were based on DNA, called DNA Vaccine, and were initiated during 2000s. Such approaches were tested and found to be successful. In DNA vaccine, infused nucleoprotein (NP) or glycoprotein (GP) of EBOV acts as an antigen to activate host immune responses and infused antigen-delivering DNA boosts the immunization [49]. Recently, a trial using Vesicular Stomatitis Virus (VSV) platform has been devised as bivalent vaccine against EBOV showing systemic immune responses safe-guarding animals after injection [50]. This non-segmented, negative stranded RNA virus is likewise celebrated to be a superlative competitor for recombinant DNA vaccine belvedere across several filoviruses as the virtue of the virus is an animal pathogen that does not coax any kind of serious manifestations in humans [50,51].VSV based candidate vaccines (VSV-GP) have been successful in mice and have proved to give complete protection and also, have shown to be effective in the post exposure treatment regime. Since VSV-based vaccines are replication competent recombinant viruses, their use have been suspected in humans [88]. VSV based vaccines are an exclusive feature that is very precious in laboratory accidents, outbreak and bio-weapon exposure situations [52]. A review on phase-I trials of EBOV vaccines can be in found in Lambe et al. [53].

Antibodies based therapy
Human survivors of EVD are liable to early accumulate long standing neutralizing antibodies (NAbs) which may bind to EBOV structural enveloped glycoprotein (GP) [54]. Identifying such NAbs and their mechanism of activity is important for the development of novel immunotherapy and vaccines against EBOV. The broadness of aegis of such NAbs is wavering and still enduring study, which includes use of Convalescent Blood Products (Plasma) and ZMapp. Convalescent Plasma therapy is an immunotherapy, whereas ZMapp is a monoclonal antibody which degrades the EBOV transmembrane glycoprotein, helping the virus to attach, fuse and enter the host organism [55].

Under-sized molecules and drugs
There are numerous known and accepted abettors and pin-pointed compounds that show paradoxical-EBOV activity. For instance, benzodiazepine derivative which forestalls EBOV entry into the host cells [56]. We have matured drugs against EBOV, which have proved to be competent, like Favipiravir. Favipiravir performs antiviral activities against negative stranded RNA viruses, and has been administered in European Centres in the last EVD outbreak. Molecules such as Phosphorodiamidate Morpholino Oligomers (PMOs), and small interfering RNAs (siRNAs) are found useful in abbreviating carnage when supervised to NHPs up to 1 h after disclosure [57,58]. AVI 6002 is a unification of positively charged PMOs devised to point mRNA sequences of VP24 and VP35 in EBOV. It is presently going through phase-I clinical trials for EBOV post-exposure treatment [39]. Brincidofovir (CMX001) is a prodrug of cidofovir and is an emphatic anti-DNA antiviral antidote by impeding viral replication, discouraging viral DNA polymerases. Currently, it is going through Phase-III clinical trials for its application against adenovirus and cytomegalovirus. Hence, Brincidofovir are used in crunch positions in subjects vitiated with EBOV [59]. BCX4430, another antiviral, restrains viral RNA polymerase activity circumlocutory through non-obligate RNA chain termination, exhibiting adequacy in pre-exposure EBOV treatment in vitro in small animal model. It showed protection from antagonist EBOV after taking BD oral and IM administration for 9days [60]. However, combination of ZMapp and high dosage of Favipiravir have proven to yield better results than the rest.

Resistance or Resilience: what is better for combating Ebola?
In ecology, resistance refers to the characteristic feature of populations (communities), which remain unchanged when subjected to any turbulent episode, both natural and artificial (man-made). On the other hand, resilience is defined as the potential of the populations (communities) to respond to the disturbances by resisting the damage caused and by rapid recovery. If we correlate the two terms, we may answer how communities have coped EVD. Resilience in general means "Elasticity", i.e., transforming our medical health systems into resilient ones in order to become multifaceted, giving care and support to the patients to overcome the past and prepare them for a better future [61,62]. Healthcare systems can be termed as resilient only when they have the zeal and springiness in their mode of action towards such epidemic disease spills [63]. They must also provide patients with everyday benefits and optimistic results for living an exceptionally beautiful future. This joy of double benefit and an upgraded performance in all times is what is known as "the resilience dividend" [64].
Resistance in disease spill-over is rarely observed. Resilience, on the other hand, proves to be a better option. The Ebola outbreak in the past has shown us a mirror that we lag behind when it comes to a resilient human healthcare system. Constructing resilience is ambience-dependent and verbose which needs progressive appraisal of healthcare systems, strengths and weaknesses, contributions in susceptible and sensitive peripherals of the system ahead of any cataclysmic episodes, support during such emergencies, and an analysis and audit of the conduct of work after the outbreak. Resilience is not a passive or stagnant construct, for instance, the accelerated measures of improvement from a disaster is a fundamental and an essential part of accomplishment [64].
Resilient Heath System (RHS) is a comprehensive and a universal civic congenial approach which requires concerted and a corporate acknowledgement from all over the world. Capitalizing for this antiphon can materialize from conventional subdued donor sources, or from international sources where countries contribute a hefty amount [65]. Diversified fields such as-ecology, engineering, complex adaptive systems, psychology, and civil health have created resilience scheme [66,67]. Here we propose five mantras to a RHS, composed of five rules that must be followed by every medical healthcare system, as depicted and described in Fig. 8. Along with the therapies mentioned in previous section, EVD patient needs a special yet specified care. Clinical researches can be integrated into standard care towards the evaluation of EBOV specific therapies [68]. Optimized Standard Of Care (oSOC) is focused on the standardized routine of care, which involves 6 steps [68], depicted in Fig. 9, which is self-explanatory.

Ebola mathematical model with control
Optimal control theory has been applied to study the dynamic mechanism of EBOV, which can be used to reduce its susceptibility in the population. The mathematical model of EBOV was created by Bonyah and collaborators [69], which provides EBOV control strategy. In this mode, two treatment controls are applied to the infected and late stage infected (super) human population. For this, Pontryagin's Maximum Principle [70] is employed to characterize optimally control, which is then solved numerically. It is observed that time optimal control exist in the EBOV model. The activation of each control showed a positive reduction of infection. The overall effect of activation of all the controls simultaneously reduced the effort required for the reduction of the infection quickly [69].
In Bonyah et al. [69], mathematical model of EBOV disease include prevention and treatments measures as optimal control. By exploring and using Pontryagin's maximum principle, condition for optimal control to minimize disease in a finite time can be derived and analyzed. The model system of Ebola has six NLDEs (Non-Linear Ordinary Differential Equations), which have been given by [70] and Bonyah et al. [69], as follows: Given all S(0), E(0), I 1 (0), I 2 (0), F(0), and R(0) > = 0, where the model partitions total populations, denoted by epidemiological sub population of susceptible (S), those exposed to Ebola virus (E), those individuals with the first stage of infection (I 1 ), those individuals with the second stage of infection(I 2 ), and the fraction of the population who recovered are denoted by (R). The fraction of population who died, and/or being processed for burial is represented as F. Coefficients β 1 , β 2 and β f are the rates at which the susceptible become infected by an infectious individual in the first, second, and burial stage per unit of time, respectively. An exposed individual moves to the first stage of infectious class at a rate of α. The average length of first stage of illness is denoted by γ 1 −1 and the average length of second stage of illness is . δ 1 denotes the fraction of infected who progress to the second stage of infection and δ 2 is the fraction who subsequently progress to death. The natural per capita mortality of EVD rate is denoted by μ. Fig. 10 presents a schematic diagram as compartment of Ebola Model, where population of susceptible (S) may be exposed to EBOV (E), which can further go to stage 1 (I 1 ) and may either recover (R) if treated properly, or moved to stage 2 (I 2 ). From stage 2 (I 2 ), patient may either be recovered (R), or died (F). The control functions u 1 (t), u 2 (t), and u 3 (t) are bounded Lebesques integrable functions. The objective is to reduce the number of infected individuals with the EBOV, while at the same time keeping the cost of treatment very low [69].The optimal control strategy applied to this model shows that proper medication on the right time basis will ensure that EVD can be cured [69].

Challenges ahead
Ebola has been proven to be the most dangerous and fatal disease to mankind since its first outbreak in 1976. The disease transmission is known to be uncontrolled, and it is probable for its spill over to the unaffected areas of the world. The Centre for Disease Control and Prevention (CDC) is engaged extensively with allies to help stop the epidemic at its source in Africa. Every month, thousands of people from affected areas travel to different part of the world including other parts of Africa, United States, Europe, and Asia. As long as Ebola is spreading in these regions, clinicians are required to be vigilant to the likelihood of EVD, assess travel history, and immediately separate and test ill travelers who have travelled toEVD affected regions in the past 3 weeks, and have manifestations persistent with EVD [71]. The CDC has circulated guidelines about identifying, separating, diagnosing, and treating patients (http://www.cdc.gov/vhf/ebola/hcp/infectionprevention-and-control-recommendations.html). Some of the major challenges in Ebola research are as follows:

Directions for management of EVD
A large number of healthcare workers need to be trained to manage severe and fatal EVD. It may also be planned to treatment post-EVD complications, including social and psychological support. Although, several governmental and non-governmental agencies have been established to control the activities related to any outbreak, and establishment of supportive health-care system, but still basic challenge is that survivors need some specialised services. Therefore, mental health, eye care, and professionals need to be trained, and clinics need to be established [21].

Setting sight on targeting the virulent pathogen-contingencies and challenges
Remedial treatment for viral contagion can be universally assorted as agents that intrude the virus and its replication cycle without any deviation, or as abettors that aid and entrench host immune deterrence. According to the golden rule, there are bountiful targets and multifarious plans for both the above mentioned categories. This exuberant of quiescent targets could result in numerous remedial approaches. In conjunction with antisense targeting of the viral genome, inhibition of the replicase or polymerase activity by small-molecule inhibitors, including other specific molecular targets are important for the formation of a replication-competent complex [72].The modernistic advancement of reverse genetics and filovirus reporter-based mini-genomes [73], conjointly, Green Fluorescent Protein (GFP)expressing EBOV [74], is likely to promote the apperception of inhibitors of filovirus replication.

Ancillary therapeutics for Ebola
Filovirus infections are correlated with many pathological circumstances, inclusive of promulgated intravascular coagulation, that has given results from up-regulation of tissue factor on the surface of leukocytes [75]. Limited accomplishment contra to EBOV bugs in rhesus monkeys applying recombinant nematode anticoagulant protein C2 has lately been proclaimed [76]. Despite the fact, the study is promising, the service of anticoagulant therapy in humans which needs  may be exposed to EBOV (E), which can further go to stage 1 (I 1 ). From I 1 , it may either recover (R) if treated properly, or moved to stage 2 (I 2 ). From stage 2 (I 2 ), patient may either be recovered (R), or died (F) (Source: [69] supplementary studies distinctly in consolidation with clear-cut antiviral remedies [77].

Aggrandizing elemental immunity
The ambition of a few antiviral abettors is to tip antithesis of the immune response facing innate immunity and avows definite immune consent mechanisms, viz. adaptive immunity, to ratify [7]. At the crisis of copious native (elemental), immune responses are interferons, a family of molecules that can precisely elicits antiviral responses. Though, the services of interferons as broad-spectrum antivirals antiquate finite both by the ephemerality and lethality of their effects. This has threatened attention about the expectancies for a wide array of other newly characterized cytokines that also encourage innate immunity. Furthermore, other conveniences for drug interference have emerged in targeting viral pathogens. The recognition of proteins originated by vaccination and influenza virus which serve as interferon antagonists [78,79] was ensued by presentation that Ebola viruses [80] and Marburg viruses [81] also produce interferon antagonists.

Is Ebola the new bio-weapon?
Bioterrorism and Bio-weapons are the new tinge of today's demagogic society. The discombobulating caused by the incendiary communities can be anything, of any form. The spill-over of virulent bugs put a perpetual impendence to the global health. Enough heed is paid to the emanation of comparatively unfamiliar or unknown bugs, viruses etc, e.g. EBOV. Most epidemics materialize due to external and topographical factors. Occasionally, notwithstanding, hominid prying with nature impacts lead to the spill-over of the diseases. A few zoonoses plunge to human host because the rainforest habitat of former animal hosts is reduced. The success of bio-terroristic effort is measured by societal disruption and panic, and not by abrupt number of casualties [82]. Among three classes of bioterrorism agents defined by CDC, category A includes the highest-prerogative abettor which can be easily transmitted from person-to-person causing secondary and tertiary cases. It causes high fatality and high impact on public health, public panic and social disruption. Filoviridae have been placed in category A, and EBOV also causes lethalness, may be misused as an ultimate destructive agent for mankind.

Lesson learn from past outbreaks
Modern lessons learnt from Ebola outbreaks in the past should be standardized. The previous experiences of malfunctioning and failure have to be remembered and accordingly new practices have to be implemented [83]. The basic lessons that have been learnt seeing the past outbreaks are as follows [84]: Disease detection and disease response systems must be invigorated. International funding must be present beforehand. International stocks of protective apparatus and supplies must be controlled and its uses must be available.
The public health society must identify the positive potential of the international media in organizing public ideas and resources for infectious disease control, and they must entertain their needs without backing off from the primary mission.
It becomes an utmost responsibility of WHO to educate people round the globe about any new outbreaks of diseases by utilizing electronic communication systems.
Challenges that still lie ahead in eliminating EBOV There is an urgent need to accelerate the rate of drug and vaccine development so as to prevent the spread of the disease, and to eliminate it. Ethical issues have been raised for usage of experimental treatments and vaccines which are in very limited supply. However, a vaccine which is safe and effective would further protect in outbreak situations [71].
Candidate vaccines should be evaluated and updated on a regular basis.
Novel technologies must be quickly adopted, as they are very likely to yield faster and better results. These technologies and analytical equipment must be inexpensive so that any small laboratory can also work with them.
A holistic approach should be used in order to understand the mechanism and treat the atrocious EVD.
In order to understand the mechanism and the dynamics of the virus, Systems Biology, Computational, Analytical, Mathematical fields of studies must be amalgamated, which eventually would lead to the formation of framework, describing the intrinsic details of Ebola.
There is a need to strengthen our health and security agendas, to fight with the virulent pathogens.
More and more consultants, nurses, are needed who are willing to treat the Ebola patients. There should be better hospital infrastructures, and clinics, which are fully fuelled with the laboratories and technologies.
Ebola outbreak also comes with a chance to scale up mental healthcare sorely needed within the affected countries. It is very important to provide heed to the mental health being [85].

Conclusion
Ebola Virus Disease (EVD) is a potentially threatening illness to human health globally, caused by Ebola Virus (EBOV) of the virus family Filoviridae. EBOV is an enveloped virus composed of 18.8-19 kb single-stranded, negative sense RNA genome that encodes 7 proteins and has surface glycoprotein presenting roles in cell attachment, fusion and cell entry involved in immune evasion and pathogenesis of disease. Transmission of the virus is generally zoonosis which involves animals and humans. As it was identified that fruit bats are the reservoir hosts of the Ebola Virus and therefore they carried this virus and transmits to other animals and further to humans. The spill-over of infections basically occurs from bats to humans but the dynamical mechanism of EBOV is understood by 3 factors, namely, Biological Factors, Environmental Factors and Social Factors, and therefore, the infectious interaction between bats and the ecosystem is responsible for the spillover dynamics of deadly diseases either directly (bats -> humans) or indirectly (bats -> animals -> humans). So, to understand the framework for the holistic study of disease dynamics mechanism of EBOV, various approaches such as Life Sciences, Mathematics, Systems Biology, Computational, and Analytical methods need to be applied.
The Ebola outbreak in the past has shown us a mirror that we dawdle behind when it comes to a resilient human healthcare system. Constructing resilience is ambience-dependent and bombastic which needs progressive appraisal of healthcare systems, strengths and weaknesses, contributions in susceptible and sensitive components of the system ahead of any cataclysmic episodes. Resilient Heath System (RHS) is a global and a universal civic congenial approach which requires concerted and a corporate acknowledgement from all over the world.
There is a big challenge in the treatment of EVD as there is no approved therapeutics so far. But some of the diagnostic methods such as ELISA and rRT-PCR are generally used. In addition, a number of vaccines and antiviral drugs are under the development against EVD by using animal models such as mice, guinea pigs, and NHPs to test the safety and efficacy of the vaccines or drugs, which gives results for only phase I clinical trials. Phase II, III, and IV trials didn't give better results due to lack of equipment, resources and technology. Various small molecules and antiviral drugs, including as Favipiravir, siRNAs, PMOs, AVI6002, Brincidofovir, BCX4430, and ZMapp, have been developed. Among these, the most promising and active antiviral drugs are ZMapp and Favipiravir. Hence, to get a victory against Ebola, we still require powerful drugs and vaccines. In addition to therapeutics and medications, optimized care using resilient health system is very important. Optimal control theory was used to develop dynamic mathematical model of EVD which can be used to reduce the sensitivity in the population. The Centre for Disease Control and Prevention is engrossed broadly with allies to help stop the epidemic at its source in Africa. Thousands of people from affected areas travel to different part of the world. As long as Ebola is growing, practitioners are required to be very alert to the probability of EVD, check travel history, and immediately separate and test ill travelers who have travelled to EVD affected regions in the past 3 weeks [71]. However, it is a challenging task. Regardless of the fact, the service of anticoagulant therapy in humans needs additional studies distinctly in confining with clear-cut antiviral remedies. Though, the services of interferons as broad-spectrum antivirals archaize finite both by the brevity and fatality of their effects. This has jeopardized attention about the expectancies for a wide array of other newly characterized cytokines that also inspires innate immunity. To elevate the predictability, it becomes mandatory to identify infected persons as soon as possible, which requires latest diagnostic techniques and treatment facilities. A single nonchalant activity can be disastrous. By following precautionary measures, spread of the disease can be restricted to a large extent. Challenges in combating Ebola are not only limited to resources and knowledge, but also public and personal hygiene that must be adopted in the society.

Disclosures
The authors declare that there is any conflict of interest in the publication of this manuscript.


s2fieldsofstudy: Biology Medicine

text:
Transmissible endoplasmic reticulum stress from myocardiocytes to macrophages is pivotal for the pathogenesis of CVB3-induced viral myocarditis

Infiltrating macrophages have been proven as a pivotal pathological inflammatory cell subset in coxsackievirus B3 (CVB3) induced viral myocarditis. However, the mechanisms underlying the initiation and promotion of macrophage pro-inflammatory responses are still blur. We previously reported that cardiac ER stress contributed to CVB3-induced myocarditis by augmenting inflammation. In this study, we focused on the influence of ER stress on the macrophage inflammatory responses in the viral myocarditis. We found that ER stress was robustly induced in the cardiac infiltrating macrophages from CVB3-infected mice, and robustly facilitated the production of pro-inflammatory cytokines (IL-6, IL-12, MCP-1 and IP-10). Consistently, adoptive transfer of ER stressed macrophages significantly worsened the viral myocarditis; while transfer of ER stress-inhibited macrophages obviously alleviated the myocarditis. To our surprise, this significantly activated ER stress was not directly caused by the virus stimulation, but was transferred from the CVB3-infected, ER stressed myocardiocytes via soluble molecules in a TLR2, 4-independent way. In the present study, we reported that the transmissible ER stress from the infected myocardiocytes to macrophages could augment the pro-inflammatory responses and promoted the pathogenesis of viral myocarditis. Blocking ER stress transmission, instead of inhibiting its initiation, may represent novel therapeutic strategies against viral myocarditis.

Endoplasmic reticulum (ER) is an essential organelle responsible for protein synthesis, folding, and modification. Stimuli perturbing ER homeostatic would cause stress and activate the subsequent unfolded protein response (UPR) consisting of three highly coordinated signaling cascades: IRE1α (inositol-requiring 1α ), PERK (PKR-like ER kinase) and ATF6 (activating transcription factor 6) 16,17 . Upon activation, these three ER stress sensors would disassociate from 78 kD aglucose-regulated protein (GRP78) and modulate the down-stream transcriptional and translational machineries. Recently, a line of evidence revealed the crosstalk between ER stress and tissue inflammation. Kaser A and colleagues 18 reported that depletion of X-box binding protein 1 (XBP1), a key transcription factor in IRE1-associated pathway, in epithelial cells led to the spontaneous enteritis and increased susceptibility to colitis. In addition to impacting tissue cells, ER stress also directly manipulates immune cell activities. For example, in combination with toll-like receptor (TLR) agonists, ER stress can significantly increase the production of IL-23 by dendritic cells 19 , remarkably enhance the production of inflammatory cytokine (IL-6, IL-23) by macrophages 20 and drive the processing of pro-IL-1β in response to LPS stimulation 21 . In addition, ER stress is also responsible for the differentiation of alternatively activated macrophages in the context of atherosclerosis 22 . Considering that cardiac ER stress substantially contributes to viral myocarditis by augmenting inflammation and promoting myocardiocytes apoptosis 23,24 , and infiltrating macrophages are critical pathological inflammatory cells in viral myocarditis 11,15,25 , herein we hypothesized that ER stress might occur in the cardiac infiltrating macrophages in the context of CVB3 myocarditis and impact the pro-inflammatory responses of macrophages.
In this study, we showed that ER stress was robustly activated in the cardiac infiltrating macrophages from CVB3-infected mice, and obviously promoted the production of pro-inflammatory cytokines (IL-6, IL-12, MCP-1 and IP-10). Adoptive transfer of ER stressed macrophages to CVB3-infected mice significantly aggravated the viral myocarditis. Interestingly, we found that this potent macrophages ER stress was not directly caused by CVB3 stimulation, but was transferred from the CVB3-infected, ER-stressed myocardiocytes via soluble molecules. Take together, our study suggested that the transmissible ER stress from CVB3-infected myocardiocytes endowed the cardiac infiltrating macrophages with pro-inflammatory properties, and contributed greatly to the pathogenesis of viral myocarditis.

Results
Cardiac ER stress significantly aggravated CVB3-induced viral myocarditis. Mice were infected with 10 3 plaque-forming unit (PFU) CVB3 at day 0 and the induction of cardiac ER stress was monitored at day 7 by detecting the expression of its specific markers GRP78 and GRP94. As shown in Fig. 1A,B, obviously increased GRP78 and GRP94 were seen in the hearts of CVB3-infected mice compared with the control group, indicating the robust induction of cardiac ER stress. To further elucidate the role of ER stress in the development of viral myocarditis, CVB3-infected mice were treated with 100 mg/kg ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or 2 mg/kg activator tunicamycin (Tm) at day 1 and day 4 post infection, and the expression of GRP78 and GRP94 in mice heart tissue were detected by real-time PCR (Fig. 1F,G), and then the changes in myocarditis severity were determined. It was found that compared with the control infected group, mice treated with TUDCA showed significantly reduced CK-MB activity (590 vs 967, Fig. 1H), relieved myocardial inflammation (Fig. 1I,J), an increased the survival rate (90% vs 40%, Fig. 1K) and obviously improved left heart ejection fraction (LVEF) and left heart fractional shortening (LVFS) (Fig. 1L,M). And mice systemic hemodynamic parameters were evaluated by the indices of systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR). As shown in Fig. 1N,O,P, mice treated with TUDCA showed significantly higher SBP (102.7 vs 89.3, P < 0.001) and lower DBP (40.5 vs 47, P < 0.01) than infected control mice. All these data indicated the remission of viral myocarditis. In contrast, mice treated with Tm exhibited substantially aggravated myocarditis ( Fig. 1I-P).

ER stressed cardiac macrophages played a pivotal pathological role in CVB3-induced myocarditis.
As the earliest infiltrating and predominant inflammatory cells, cardiac macrophages have been proven to contribute greatly to the development of viral myocarditis, however, it is still unclear whether the cardiac ER stress could influence the inflammatory responses of infiltrating macrophages. Here, we detected the dynamic activation of ER stress in the cardiac infiltrating macrophages from CVB3 infected mice, and found that macrophages began to undergo obvious ER stress even at the very beginning of enrichment in heart tissues (day 3 post infection, Fig. 2A,B) 11 . As time passed by, the extent of macrophage ER stress was further increased, and reached the peak at day 5 and remained the plateaus level at day 7. Of note, these dynamic changes of macrophage ER stress were consistent with the progress and magnitude of viral myocarditis 11 . The results were further confirmed by the study of in situ immunofluorescence, which showed that cardiac GRP78 expression was obviously induced after CVB3 infection, confirmed that ER stress was efficiently induced in the CVB3-infected hearts. More importantly, F4/80 + cells co-localized with the expression of ER stress marker GRP78, indicating that infiltrating macrophages underwent ER stress (Fig. 2C). Interestingly, inhibition of the ER stress in the cardiac macrophages could potentially decrease the production of pro-inflammatory cytokines (IL-6, IL-12, MCP-1 and IP-10) (Fig. 2D), indicating that ER stress amplified the pro-inflammatory responses of cardiac macrophages. To further validate the role of ER stressed macrophages in viral myocarditis, ER-stressed or ER stress-inhibited bone marrow-derived macrophages (BMDMs) were prepared respectively, and adoptively transferred into the macrophage-depleted recipients at day 3 post CVB3 infection. It was found that compared with the control mice receiving BMDMs, mice receiving ER stressed BMDMs succumbed to more severe viral myocarditis, as reflected by the more body weight loss, increased serum CK-MB, exacerbated pathological changes and a lower survival rate (Fig. 3). Conversely, mice receiving ER stress-inhibited BMDMs exhibited obviously alleviated myocarditis (Fig. 3). All these data revealed that ER stressed macrophages exerted potent pathological effects on CVB3-induced viral myocarditis. Macrophage ER stress was not directly induced by CVB3 stimulation, but was transferred from CVB3-infected, ER stressed myocardiocytes. Since ER stressed macrophages took part in the pathogenesis of viral myocarditis, it is important to decipher the initiation mechanism of macrophage ER stress. It was found that in vitro stimulation of murine macrophage cell line RAW264.7 cells with CVB3 at various doses (ranging from 1 to 100 multiplicity of infection (MOI)) could not induce obvious ER stress responses (Fig. 4A). Even extending the CVB3-stimulation time scale to 24 h, no obviously up-regulated GRP78 was observed either in RAW264.7 cells (Fig. 4B) or in BMDMs (Fig. 4C). In contrast, these macrophages underwent robust ER stress in response to Tm treatment. We also stimulated splenic macrophages of naive mice with CVB3 in vitro, no detectable UPR activation was detected (Fig. 4D), suggesting that CVB3 couldn't infect macrophages. All these data suggested that macrophage ER stress was not directly initiated by virus stimulation, but by other factors. Heart sections from shaminfected mice (upper panels) or CVB3-infected mice (lower panels) were stained with Cy3-labeled rabbit antimouse GRP78 (red) or FITC-labeled rat anti-mouse F4/80 (green), and DAPI identifying the nucleus (blue). Images are representative of at least 3 independent determinations. (D) Splenic macrophages were isolated at day 7 post infection and the expression of GRP78 was detected by real-time PCR. (E) Cardiac macrophages were isolated at day 7 post infection and treated with TUDCA, the production of pro-inflammatory cytokines (IL-6, IL-12, MCP-1 and IP-10) was determined by real-time PCR. Each group contained 5-6 mice. Individual experiment was conducted 3 times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.
As previous literature showed that in the context of tumor microenvironment, ER stress could be transferred from tumor cells to macrophages, and then endowed the latter with a pro-inflammatory phenotype 20 . Inspired by this study, we assumed that ER stress in the cardiac macrophages might be also transferred from the surrounding tissue cells (myocardiocytes) in the condition of CVB3 myocarditis. To testify this hypothesis, we dynamically detected the myocardiocyte ER stress in the infected mice, and found that the expression of GRP78 and GRP94 appeared to increase (about 2-fold) at day 1 post infection and peaked at day 3 (about 10-and 16-fold, respectively), but then robustly decreased at day 5 and day 7 (Fig. 5A,B), suggesting that the infected myocardiocytes underwent ER stress. In line with these in vivo data, in vitro infection of freshly isolated neonatal myocardiocytes also substantially induced ER stress (Fig. 5C), and this effect seemed dependent on the infectivity of CVB3 virus, as it was completely deprived once CVB3 was inactivated (Fig. 5D).
To determine whether ER stress could be transferred from the infected myocardiocytes to the infiltrating macrophages, macrophages were co-cultured with the infected myocardiocytes for 24 h, and then the induction of ER stress was determined. As shown in Fig. 6, compared with the control macrophages, macrophages co-cultured with the CVB3-infected myocardiocytes or Tm-treated myocardiocytes expressed much higher levels of GRP78, phospharylated IRE1α , eIF2α and PERK, and obviously ATF6 cleavage. In addition, we also detected expression of the spliced form of Xbox-binding protein 1(XBP1s) mRNA by RT-PCR, the data showed that the spliced XBP1 significantly upregulated in the macrophages (RAW264.7 cell lines and BMDMs) co-cultured with CVB3-infected myocardiocytes in comparison to the counterparts co-cultured with non-infected myocardiocytes (Fig. 6K,L). All these data indicated the efficient induction of all of the three UPR arms. These data revealed that macrophage ER stress could be transferred from CVB3-infected, ER stressed myocardiocytes.

ER stress transmission from the infected myocardiocytes to macrophages was mainly mediated by soluble molecules, but not cell-cell contact way.
To determine whether cell-cell contact was required for this ER stress transmission, infected myocardiocytes and macrophages were co-cultured on the opposite sides of a porous transwell membrane insert. As illustrated in Fig. 7A, disrupting cell-cell contact did not obviously impede ER stress transmission between the co-cultured infected myocardiocytes and macrophages, Figure 3. ER stressed macrophages obviously aggravated CVB3-induced myocarditis. After depleting macrophages, mice were infected CVB3 at day 0 and received Tm-treated BMDMs, TUDCA-treated BMDMs, BMDMs or PBS at day 3, and the severity of viral myocarditis was monitored by body weight loss (A), serum CK-MB activity (B), percentage of myocardial inflammation (C), pathological observation of heart tissues (D) and survival rates (E). CVB3-infected mice without macrophage depletion were applied as a control group. Each group contained 4-5 mice. For survival rate evaluation, each group contained 10 mice. Individual experiment was conducted 3 times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.
suggesting that soluble molecules in the conditioned medium were mainly responsible for this ER stress transmission. These results were further confirmed by the efficient induction of ER stress in the macrophages co-cultured with the infected myocardiocyte-derived conditioned medium. It was founded that GRP78 protein appeared to be up-regulated at as early as 12 h post co-culture and further increased at 24 h (Fig. 7B). We further investigated the ER stress-transferring abilities of various conditioned medium derived from the infected myocardiocytes, which was collected at different time-points (6, 12 and 24 h) post infection, and found that only the conditioned medium collected at 12 and 24 h possessed this ER stress-transferring ability, while the medium collected at 6 h did not. These results were consistent with the dynamics of ER stress induction in the CVB3-infected myocardiocytes, in which GRP78 was obviously up-regulated at 12 h, but not 6 h post infection (Fig. 5C). Taken together, these data indicated that the conditioned medium, but not cell-cell contact, was operative in the ER stress transmission between the infected myocardiocytes and macrophages. Transferred ER stress robustly amplified the production of pro-inflammatory cytokines. We next detected whether this transmissible ER stress could impact the pro-inflammatory property of macrophages, and found that compared with the control cells co-cultured with the uninfected myocardiocyte-derived conditioned medium, macrophages co-cultured with the infected myocardiocyte-derived conditioned medium produced much higher levels of IL-6, IL-12, MCP-1 and IP-10. Consistently, treating co-cultured macrophages with TUDCA could robustly decrease the production of these pro-inflammatory cytokines (Fig. 8), further confirming the inflammation-promoting effect of the transmissible ER stress. These results were also consistent with the in vivo data as shown in Figs 2C and 3, in which ER stress facilitated the pro-inflammatory phenotypes of cardiac infiltrating macrophages and contributed to the development of CVB3-induced viral myocarditis.
ER stress was transferred from infected myocardiocytes to macrophages in a TLR2/4-independent way. Considering that TLR4 signaling pathway has been proven to be involved in the ER stress transmission from tumor cells to macrophages 20 , therefore herein we explored the roles of TLR2 and TLR4 in the ER stress transmission between infected myocardiocytes and macrophages. After co-culturing BMDMs derived from TLR2 −/− or TLR4 −/− mice with uninfected, CVB3-infected or Tm-treated myocardiocytes for 24 h, the GRP78 expression in these co-cultured macrophages was determined by western blot assays. As shown in Fig. 9, GPR78 expression was significantly up-regulated not only in wild type macrophages, but also in TLR2 −/− and TLR4 −/− macrophages, indicating that this transmission of ER stress was independent on TLR2 or TLR4 pathways.

Discussion
ER stress generally triggered by accumulated mis-folded proteins was initially shaped as a serial of host compensatory responses to adapt and recover ER homeostasis. If the stress is persistent and strong, ER stress-associated apoptosis signaling pathways will be activated. In the context of CVB3 infection, ER stress-mediated apoptosis has been evidenced 24 . In addition to its effect on cell survival, ER stress also possesses immune modulation capability and participates in many infectious, metabolic and immune pathological diseases [26][27][28][29] . It not only facilitates the activation of innate immunity 30 , promotes pro-inflammatory cytokine production 31 , but also contributes to macrophage polarization as well as foam cell formation 22 . Since ER stress aggravates CVB3-induced viral myocarditis 23 , and macrophages represent one of the most important inflammatory cell subset in CVB3-infected mice, herein we emphatically studied the initiation mechanism of macrophage ER stress as well as its impact on the process of viral myocarditis.
It was found that the cardiac infiltrating macrophages underwent potent ER stress at day 3 post CVB3 infection, and continued to increase as disease progressed. When inhibiting ER stress, the production of pathological pro-inflammatory cytokines by the cardiac infiltrating macrophages was substantially reduced, indicating the blunted pro-inflammatory phenotype of macrophages. The pathological role of ER stressed macrophages in CVB3-induced myocarditis was further confirmed by the adoptive transfer of Tm pre-treated macrophages into the macrophage-depleted recipient mice at day 3 post infection.
Next, we tried to elucidate the trigger of macrophage ER stress in CVB3-induces myocarditis, and surprisingly found that the virus stimulation could not directly induce macrophage ER stress. Then we detected whether CVB3 could infect macrophages, both of our and other groups 32 observed that although CVB3 virus particles can enter cells (Fig. 5E), but they can not efficiently replicate there (as shown by the limited amount of positive-and negative-strand RNAs of CVB3, Fig. 5F,G), so the amount of released virus particles is very limited (Fig. 5H). This deficiency in supporting virus replication might account for the macrophage resistance for CVB3-induced UPR. In other words, the activation of CVB3-induced UPR might depend on abundant intracellular production of virus replication or abundant amount of viral load. Therefore, the ER stress of macrophages in CVB3-infected mice hearts was more likely to be launched by the cardiac microenvironment factors, but not by virus alone. In support of our hypothesis, Mahadevan NR et al. 20 demonstrated that ER stress could be transferred from tumor cells to macrophages, and promoted the production of pro-inflammatory cytokines. In this study, we did observe that the myocardiocyte ER stress was induced at as early as day 1 post CVB3 infection and peaked at day 3, however, it dramatically decreased at day 5 and day 7. This dynamic was inconsistent with the process of myocarditis in which cardiac inflammation and injury begin to appear at day 3, and peak at day 7, indicating that the myocardiocyte ER stress might not directly contribute to the cardiac inflammation. Of note, the peak time of myocardiocyte ER stress happened to coincide with the beginning time of macrophage enrichment in hearts, indirectly suggesting that myocardiocyte ER stress might be associated with the immune responses of macrophages. In line with these in vivo data, in vitro results also revealed that CVB3 infection could efficiently induce ER stress in myocardiocytes. Inspired by all of these, we deduced that in the condition of CVB3-induced myocarditis, ER stress in cardiac infiltrating macrophages might be transferred from infected, ER stressed myocardiocytes. As it turned out that co-culture with infected myocardiocytes could obviously induce ER stress in macrophages and significantly promote the production of pro-inflammatory cytokines. This ER stress transmission process was mainly dependent on the conditioned medium of infected myocardiocytes, suggesting that soluble molecules Figure 8. Transmissible ER stress endowed macrophages with the pro-inflammatory phenotype. RAW264.7 cells were cultured for 24 h with conditioned medium (c.m.) from infected myocardiocyte, followed by treating with or without TUDCA for 24 h, and then production of pro-inflammatory cytokines (IL-6, IL-12, MCP-1 and IP-10) was detected by real-time PCR assays. Individual experiment was conducted 3 times with similar results. **P < 0.01, ***P < 0.001. Figure 9. ER stress transmission between infected myocardiocytes and macrophages was independent on TLR2/4 pathways. BMDMs derived from wild type (WT), TLR2 −/− or TLR4 −/− mice were cultured with conditioned medium (c.m.) from uninfected, CVB3-infected or Tm-treated myocardiocytes for 24 h, and then expression of GRP78 in co-cultured macrophages was detected by western blot assays. (A) Expression of GRP78 protein in co-cultured macrophages was detected by western blot assays (B) Semi-quantification of co-cultured macrophages GRP78 expression. Individual experiment was conducted 3 times with similar results. *P < 0.05, **P < 0.01.
were responsible for this transmission. Our study was further supported by the previous study which showed the conditioned medium-mediated transmission of ER stress between tumor cells and macrophages 20 . In this study, we further extended this ER stress transmission phenomenon from tumor diseases to the field of infectious and cardiovascular diseases. Our data were further supported by the observation that CVB3 infection up-regulated the soluble damage associated molecular pattern HMGB1 (High mobility group box 1 protein) 33 , which was also reported as an ER stress inducer 34 . Besides of increased GRP78 in the cardiac macrophages, we also found that GRP78 expression in splenic macrophages was significantly up-regulated in infected mice compared with the control mice (Fig. 2E), indicating that UPR activation could also be induced in macrophages which did not infiltrated cardiac tissue after CVB3 infection. ER stress in splenic macrophages might also be transferred from other splenic tissue cells, just similar with the condition in the infected hearts.
In this study, although the detailed soluble factors involved in this transmissible ER stress are still unclear, previous studies reported that toll-like receptors (TLRs) are involved in the induction of ER stress. They could directly induce ER stress 35 and synergize with it to promote cytokine production 31,36 . More importantly, TLR4 has been reported to potentiate the transmissible ER stress from tumor cells to receiver macrophages 20 . While in our study, the transmissible ER stress seems not dependent on TLR2 or TLR4 pathway. This could be further indirectly supported by the study of Martinon and colleagues 35 , which reported that TLR4 agonists initiated the non-classical ER stress response, mainly triggering the activation of IRE1α pathway but inhibiting the ATF6 and PERK pathways. However, in our study we found that ER stress receiver macrophages underwent obvious ATF and PERK activation. This inconsistence further suggested that TLR2/4 pathway were not associated with this ER stress transmission in the context of CVB3-induced myocarditis. So far, although the exact mechanism of ER stress transmission in CVB3-induced myocarditis is still blur, we found that damage-associated molecular pattern (DAMP)-HMGB1 was robustly increased in the conditioned medium of CVB3-infected myocardiocytes (data not shown). As HMGB1 has been reported to play an important role in initiating ER stress 34,37 , it might mediate this ER stress via RAGE-mediated, instead of TLR2/4-meidated pathway. Of course, the exact role of HMGB1 needs to be further elucidated. In addition, other cytokines such as MIF (macrophage migration inhibitory factor) might also participate in this ER stress transmission. It has been reported that MIF could be induced by various virus infection [38][39][40] and function as a modulator of toll-like receptors (TLRs) signal pathways [41][42][43][44] , which were associated with ER stress induction 35 . Therefore MIF might also be very likely to associate with this transmissible ER stress between CVB3-infected myocardiocytes and macrophages.
In conclusion, our study showed that in the context of CVB3-induced myocarditis, potent ER stress responses occurred in the cardiac infiltrating macrophages and potentially augmented the inflammation. By deciphering the initiation mechanism of macrophage ER stress, we found that it was not directly caused by the virus stimulation, but was induced after co-culture with the CVB3-infected, ER stressed myocardiocytes. In addition, this ER stress transmission from myocardiocytes to receiver macrophages was mainly mediated by the conditioned medium, but not cell-cell contact way. Our study suggested that the transmissible ER stress from the infected myocardiocytes played an critical role in launching and amplifying pro-inflammatory responses of macrophages, and contributed greatly to pathogenesis of CVB3-induced myocarditis.

Methods
Mice, virus and CVB3 infection.  week-old male BALB/c mice and 1-3 day-old neonatal mice were purchased from the Experimental Animal Center of Chinese Academy of Sciences (Shanghai, P. R. China). Mice were housed in a specific pathogen-free room under controlled temperature and humidity at our institution. All animal experiments carried out in this study were conducted according to the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, People's Republic of China, 1998) and the guidelines of the Laboratory Animal Ethical Commission of Soochow University. The protocols were approved by the Ethical Committee of Soochow University. CVB3 (Nancy strain) was maintained by passage through Hela cells (ATCC number: CCL-2). Mice were infected with CVB3 by an intraperitoneal (i.p.) injection with 10 3 PFU dose of the virus.
Cell isolation, preparation and culture. BMDMs were prepared as previously described 45 . In briefly, femurs and tibias from BALB/c mice were dissected and bone marrow was flushed out. Cells were filtered through nylon mesh and plated at a density of 2 × 10 6 cells/ml in RPMI 1640 containing 30% L929 conditioned medium, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μ g/ml streptomycin for 6 days. The purity of BMDMs was > 90% as determined by FACS analysis using FITC-conjugated anti-F4/80 monoclonal antibody (BD Bioscience) and PE-conjugated anti-CD11b monoclonal antibody (BD Bioscience).
Cardiac infiltrating macrophages were prepared as previously described 11 . Neonatal mouse myocardiocytes were prepared from 1-3 day-old BALB/c mice as follows: Hearts of 8-10 neonatal mice were extracted, pooled, washed to remove blood and then minced into 0.5-1 mm 3 pieces in Ca 2+ , and Mg 2+ -free PBS. Myocardiocytes were dispersed by incubating in PBS containing 0.1% of trypsin (Sigma) and 0.05% of collagenase II (Roche) at 37 °C with constant agitation. The supernatant containing released cells was removed every 10 min into an equal volume of cold DMEM complete medium (abandon the first two times) until most of the myocardium was disrupted. Cells were collected and enriched by two sequential pre-plating steps on 90-mm dishes which removed non-myocardiocytes. Cells were incubated at 37 °C with 5% CO 2 for 48 h and then infected with CVB3 at a dose of 10 MOI.
For transwell assays, neonatal myocardiocytes (2 × 10 5 /well) were added in the upper wells (insert with 0.4 μ m pore for 12-well; BD) followed by CVB3 (10 MOI) infection, meanwhile macrophages (2 × 10 5 /well) were added in the bottom wells. Twenty-four hours later, macrophages were collected for the detection of ER stress and pro-inflammatory cytokine.
To prepare conditioned medium, 2 × 10 6 /well freshly isolated neonatal myocardiocytes were plated in 6-well plates and cultured for 48 h, and then the cell monolayers were washed 3 times with PBS and placed in 3 ml DMEM containing 10% FBS followed by CVB3 (10 MOI) infection for 6, 12 or 24 h. The supernatants were collected, centrifuged at 1,000 g for 10 min and stored at − 80 °C until using. XBP1 splicing analysis. Total RNA was extracted with TRIzol reagent (Invitrogen), reversely transcribed into cDNA with oligo dT primers (Takara, China) following the manufacturer's protocol. To evaluate relative expression levels of XBP1u/XBP1s, RT-PCR analysis was performed using PCR SuperMix (Invitrogen). XBP1 primer sequences were as follows: 5′ -AAACAGAGTAGCAGCGCAGACTGC-3′ and 5′ -GGATCTCTAAAACTAGAGGCTTGGTG-3′ , which was designed so that the PCR products contained both the spliced form of XBP1 (454-bp) and the unspliced form of Xbp1 (480-bp). GAPDH was used as a loading control, with primers as follows: 5′ -GAGCCAAACGGGTCATCATCT-3′ and 5′ -GAGGGGCCATCCACAGTCTT-3′ . The cDNA fragments were resolved on 2% agarose gels. Gels were stained with 0.5 μ g/ml EtBr in 1× TBE for 20 min, washed in water for 20 min and imaged with a gel-imaging system (Tanon). Quantification was based on relative band intensity.
Real-time PCR analysis. Total RNA was extracted with TRIzol reagent (Invitrogen), reversely transcribed into cDNA with oligo dT primers and then subjected to real-time PCR assays using a Light cycler 480 and SYBR Green system (Roche) following the manufacturer's protocol. Expression of target genes was normalized to GAPDH, and quantified using the 2 −ΔΔCt method.
Immunofluorescence assays. Mice hearts were immersion-fixed in 10% neutral-buffered formalin overnight followed by embedding in paraffin. Frozen sections about 6 μ m thick on glass slides were prepared. For antigen retrieval, the slides were exposed to hot steam for 20 min in 1 mM EDTA, pH 8.0. Mouse heart sections were incubated with a rabbit antiserum to GRP78, at 1:200 (ab108615, Abcam), and rat anti-mouse monoclonal antibody to F4/80, at 1:50 (ab16911, Abcam), at 4 °C for 16 to 18 hours. Appropriate secondary antibodies were applied for 90 minutes at room temperature. Sections were then stained with DAPI to identify nuclei. All the commercial immunochemicals were diluted as recommended by the suppliers. Images captured using the Panoramic Viewer software version 1.14.50 (3DHistech; Budapest, Hungary).
Western blot analysis. Cells were washed with cold PBS before the addition of an appropriate volume of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche). After incubation for 30 min on ice, cell lysates were centrifuged at 12,000 g for 15 min at 4 °C, and protein-containing supernatant was collected, and fractionated by 10% SDS-PAGE followed by transferring onto nitrocellulose membranes. After blocking with 5% non-fat milk-PBST, membranes were respectively incubated with primary antibodies against GRP78, PERK, phopharylated PERK, eIF2α , phopharylated eIF2α , IRE1α (Cell Signaling Technologies), phopharylated IRE1α (Thermo fisher), or ATF6 (Abcam). After washing with PBST, membranes were further incubated with an appropriate HRP-labeled goat anti-rabbit secondary antibody. Detection was carried out by enhanced chemiluminescence (Thermo Scientific Pierce) according to the manufacturer's instructions.
Adoptive Transfer of Macrophages. Macrophages of recipient mice were depleted via dichloromethylene diphosphonate (Cl 2 MDP)-loaded liposomes as described previously 11 . The depletion efficiency was > 70% as determined by FACS assays. ER stressed BMDMs were prepared by treating with 0.2 μ g/ml Tm for 12 h, and then collected and adoptively transferred to the macrophage-depleted recipients at a dose of 5 × 10 6 /mice at day 3 post CVB3 infection.
Tissue histopathology and myocarditis severity evaluation. Heart tissues were collected at day 7 post CVB3 infection, fixed, embedded in paraffin, sectioned and subjected to H&E staining. Heart sections were examined by two independent investigators in a blinded manner, and the severity of myocarditis was assessed as the percentage of the heart section with inflammation compared to the overall size of the section, with the aid of a microscope eyepiece grid as described previously 46 . Echocardiography. Mice were assessed by echocardiography system (Vevo2100, Visual Sonics, Canada) for left ventricular ejection fraction (LVEF) and left heart fractional shortening (LVFS) as previously described 47 . Hemodynamic parameters. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were determined in mice using tail cuff method (IITC Life science MRBP Blood pressure system). The blood-pressure analysis system was calibrated prior to each experiment and was programmed to measure average (from 5 consecutive readings) systolic and diastolic blood pressure and heart rate every 5 min.
Statistical analysis. All data are expressed as mean ± SEM. Statistical significance of differences in 2 groups or more than 2 groups was respectively determined by t-test or one-way ANOVA followed by Bonferroni test using GraphPad Prism version 5.0 (GraphPad Software Incorporated). P values less than 0.05 were considered to be significant.