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Recent Advances in Diagnosis, Prevention, and Treatment of Human Respiratory Syncytial Virus

Recent Advances in Diagnosis, Prevention, and Treatment of Human Respiratory Syncytial Virus Hindawi Publishing Corporation Advances in Virology Volume 2013, Article ID 595768, 26 pages http://dx.doi.org/10.1155/2013/595768 Review Article Recent Advances in Diagnosis, Prevention, and Treatment of Human Respiratory Syncytial Virus Swapnil Subhash Bawage, Pooja Munnilal Tiwari, Shreekumar Pillai, Vida Dennis, and Shree Ram Singh Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL 36104, USA Correspondence should be addressed to Shree Ram Singh; ssingh@alasu.edu Received 31 July 2013; Accepted 30 September 2013 Academic Editor: Subhash Verma Copyright © 2013 Swapnil Subhash Bawage et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Human respiratory syncytial virus (RSV) is a common cause of respiratory infection in infants and the elderly, leading to significant morbidity and mortality. eTh interdisciplinary fields, es pecially biotechnology and nanotechnology, have facilitated the development of modern detection systems for RSV. Many anti-RSV compounds like fusion inhibitors and RNAi molecules have been successful in laboratory and clinical trials. But, currently, there are no eeff ctive drugs for RSV infection even aer ft decades of research. Eeff ctive diagnosis can result in eeff ctive treatment, but the progress in both of these facets must be concurrent. The development in prevention and treatment measures for RSV is at appreciable pace, but the implementation into clinical practice still seems a challenge. This review attempts to present the promising diverse research approaches and advancements in the area of diagnosis, prevention, and treatment that contribute to RSV management. 1. Introduction Myanmar, aTh iland, Madagascar, Kenya, Zambia, Nigeria, and Columbia. The data about human RSV described in Worldwide, there are reportedly about 12 million severe literature over the years seem to have been unchanged and 3 million very severe cases of lower respiratory tract significantly, indicating the severity of RSV and the urgent infection (LRTI) in children [1]. Respiratory syncytial virus concern to address this issue. An estimate of more than 2.4 (RSV) is a common contributor of respiratory infections billion US dollars per year is the economic cost of viral lower causing bronchiolitis, pneumonia, and chronic obstructive respiratory tract infection in children [6]. pulmonary infections in people of all ages but affects mainly RSV is a Paramyxovirus belonging to the genus Pneu- children and elderly along with other viral infections leading movirus. RSV is an enveloped, nonsegmented, negative, to high mortality and morbidity [2–4]. A recent global survey single stranded linear RNA genome virus (Figure 1). RSV suggests that RSV is not prevalent throughout the year in genome (∼15 kb) has 10 genes encoding 11 proteins with the tropical regions of the globe, but the incidence peaks in twoopenreading frames of gene M2 [7, 8]. Other genes winter with a wide ranging persistence depending on the geo- include nonstructural proteins NS1 and NS2 (type I inter- graphical topology [5]. RSV has been reported to be a preva- feron inhibitors), L (RNA polymerase), N (nucleoprotein), P lent lower respiratory tract pathogen distributed worldwide (Phosphoprotein cofactor for L), M (Matrix protein), M2.1 including countries from both, the developed and developing and M2.2 (required for transcription) SH (small hydrophobic world. The major countries with RSV seasonal outbreaks protein) G (glycoprotein), and F (fusion protein). Being a include USA, Canada, Cambodia, Mexico, Uruguay, Brazil, negative strand RNA genome virus, RSV packages its own Peru,France, Finland, Norway,Sweden,Latvia, Denmark, polymerase into the nucleocapsid. Of these proteins, fusion Germany, Netherlands, Ireland, Italy, Turkey, Iran, Saudi protein (F) is indispensable for viral attachment to the host Arabia, Australia, New Zealand, China, Korea, Hong Kong, and entry into the host cell. Although the G protein is Japan, India, Pakistan, Bangladesh, Nepal, Taiwan, Vietnam, responsible for the preliminary attachment, the F protein is 2 Advances in Virology NS1 NS2 SH M2 L protein Single stranded RNA N protein P protein Matrix protein SH protein F protein G protein (a) (b) Figure 1: Structure and genome organization of respiratory syncytial virus. (a) Approximately 200 nm RSV virion particle and (b) single stranded negative RNA genome consisting of 10 genes. necessary for the fusion, budding, and spread of the virus as carriersofthe virusfor 1to3weeks. RSVinfection [9, 10]. After attachment to the host cell, RSV fuses with in children of nosocomial origin is associated with higher the host cell membrane using the F protein through the mortality than community-acquired illness because of the 6 helix coiled-coil bundle of the F protein, a mechanism pre-existing morbidity [14, 15]. Severe RSV disease risk hovers characteristically found in paramyxoviridae members [11]. forthe elderly andadultswithchronic heartorlungdisease Although the detailed mechanism of RSV infection is not or with weakened immune system [16]. RSV infection does fully understood, the most accepted mechanism is the entry not provoke lasting immunity [17] therefore, reinfection is of the nucleocapsid into the host cell mediated by the F very common [18]. Recently, RSV infection was reported to protein through clathrin mediated endocytosis [12]. The account for hospitalizations and mortality in elderly people RNA is rst fi converted into a plus strand, which serves as [19]. RSV accounted for severe lower respiratory tract infec- the template for replication; whereas for transcription, the tions including chronic lung disease, systemic comorbidities, RNA genome itself transcribes mRNA for protein synthesis and even death. At present, there is no specicfi treatment without any intermediate. for RSV infection ever since its rfi st discovery in 1956 [ 20]. Almost all children of 2 years of age will have had an RSV Currently, Food and Drug Administration (FDA) approved infection and leading to 160,000–600,000 deaths per year [4]. prophylactic drug for RSV that includes palivizumab and Approximately, 25% to 40% of infants and children at the ribavirin; administered along with symptomatic treatment first exposure to RSV have signs or symptoms of bronchiolitis drugs and supportive care. Currently, techniques used for or pneumonia. These symptoms include rhinorrhea, low- diagnosis of RSV include ELISA, direct immunouo fl rescence, grade fever, cough, and wheezing. The symptoms in adults western blot, PCR, and real-time PCR. eTh diagnosis and may include common cold, with rhinorrhea, sore throat, treatment scenario has signica fi ntly changed with the advent cough, malaise, headache, and fever. It can also lead to of advanced techniques and in-depth understanding of RSV exacerbated symptoms such as severe pneumonia in the biology, but the execution of these clinical developments elderly, especially residing in nursing homes [13]. Usually, in practice requires extensive study and time. This review children show symptoms within 4 to 6 days of infection presents the recent advances in the diagnosis, prevention, and andmostofthemrecover in 1to2weekswhile serving treatment of RSV (Figure 2). Advances in Virology 3 DNA based assays Immunoassays Nanotechnology based assays Immuno-PCR Real-time PCR ELISA Quantum dots Diagnosis LAMP LFIA Microarray PCR-ESI-MS OIA Live cells RNA imaging DFA Microarray SERS DNA vaccines Nutrition Antibodies Prevention Subunit vaccines Nanovaccines Anti-viral drugs Fusion inhibitors Antisense RNA Treatment Nanoparticles Ethnobotanicals Figure 2: A schematic representation of RSV management through coordinated diagnosis, prevention, and treatment. 2. Diagnosis also caused urticaria [34]. It is presumed to be a better bindingtargetthanthe conventional Fprotein ELISA. This There are ten known genotypes of RSV based on the sequence method could prove more eecti ff ve than the F protein analysis of the RSV genome, which suggests that the pathogen ELISA due to its higher sensitivity to the degradation of changes with time and the resulting genotypes pose a threat motavizumab. In a simple thin layer amperometric enzyme to the public health. Information from comparative genome immunoassay, RSV was detected as early as 25 minutes, at analysis has been utilized for evolutionary studies and most low cost with comparative sensitivity to that of real-time importantly for the development of detection and treatment PCR and immunou fl orescence assays [ 35]. The assay is based studies [21, 22]. Diagnostic assays for detection of pathogens on the development of a double layer sandwich method have a pivotal role in public-health monitoring [23]. However, similar to ELISA. It involves a polystyrene microarray slide diagnosis becomes difficult when the causative agent exhibits coated with monoclonal antibody which captures the antigen overlapping symptoms with other disease(s) or remains (RSV). The antigen-antibody complex is detected by horse nonsymptomatic. Hence the correct diagnosis has to be radish peroxidase conjugated secondary antibody on a screen empirically derived for the treatment. Some of the promising printed electrochemical cell coated with the substrate. molecular and biophysical techniques for RSV diagnosis are discussed below (Table 1). 2.1.2. Immunofluorescence Assay. Presently, immunou fl ores- cence is one of the most common and rapid RSV detection 2.1. Immunoassays techniques used, where the antigen is detected by a u fl o- 2.1.1. Enzyme-Linked Immunosorbent Assay. Enzyme-linked rescently tagged antibody. Direct fluorescent antibody assay immunosorbent assay (ELISA) is the enzyme facilitated col- (DFA) is a standard detection technique used for decades orimetric detection of specific protein-antibody complexes. and other RSV detection techniques are oeft n compared ELISA is extensively used for detection of various proteins at to DFA for evaluating their ecffi iency [ 24]. An indirect very low concentration in different sample types, thus making assay which uses secondary u fl orescently tagged antibody is it clinically significant in routine diagnosis of pathogens. another option to DFA. Currently, there are many molecular ELISA for RSV detection is mainly based on targeting RSV techniques that are more sensitive and reliable than DFA, F protein (antigen). Recently, several modifications of the but DFA is widely used for RSV detection in clinical samples classical ELISA technique have been ecffi iently developed due to the ease and rapidity. eTh sensitivity and specicfi ity of and employed for the detection of RSV. Sensitivity of ELISA DFA are a subject of variance as the success of the technique was increased by using the high affinity anti-RSV F antibody is dependent on numerous factors, mainly the skills of the peptides derived from the motavizumab [85]. Motavizumab technician and nature of the sample. A study showed that is a high affinity antibody based therapeutic against RSV DFA could detect RSV with a sensitivity and specificity of which binds to RSV F protein; however, it was disapproved by 77.8% and 99.6%, respectively (positive predictive value was theFDA duetohigherhypersensitivityinpatientsreceiving 98.6%and thenegativepredictivevalue was94%). DFAis motavizumab as compared to palivizumab. Motavizumab a reliable technique for RSV detection for patients tested 4 Advances in Virology ff ff fi fl fl Table 1: Comparison of RSV detection techniques. Technique Reference Principle Advantages Drawbacks Current usage status (A) Fluorescence based methods Microscopic detection of RSV with Research intent, Hospital based (1) DFA [24, 25] specific antibody conjugated with Easy procedure Human error, fading of dyes procedure, commercial fluorophore. diagnostic assays Detection of signals from fluorescent nanoparticles upon encounter with RSV Photostable, inorganic in nature, resistant Research intent (2) QDs [26–31] Toxicity, insolubility either through microscopy or ow to metabolic degradation cytometry (3) Molecular Hairpin DNA functionalized gold Probable gene silencing, beacon based [32, 33] nanoparticle with fluorophore Live cell imaging with real-time detection Research intent metabolic degradation imaging hybridization with target mRNA (B) Immunoassays Specific binding and colorimetric Easy protocol, high specificity and Cumbersome, prone to human Hospital based procedure, (1) ELISA [34, 35] detection of antigen-antibody complex sensitivity errors commercial diagnostic assays Presence of specific antigen-antibody complex formed alters the reective Needs confirmation by other Research intent, not (2) OIA [36–38] Easy, rapid, specificity, cost effective surfaces properties which is visually tests for negative samples commercialized detected Immuno-complexes detected Easy,rapid,handy,costeective, FDA Nonquantitative, limit of sample Hospital based procedure, (3) LFIA [39–42] chromatographically approved volume limits detection commercial diagnostic assays (C) Molecular methods Colorimetric/turbidimetric detection of Semiquantitative, designing Research intent, not (1) LAMP [41, 43–45] isothermal amplification of DNA using Sensitivity and specicity compatible primer set commercialized specific primer Amplification of viral cDNA and Rapid and sensitive than conventional Research intent, hospital based (2) PCR [46, 47] High limits of detection visualization of PCR product culture methods procedure (3) Real-Time Real-time amplification of target DNA or Rapid (3–5 hours), highly sensitive and Research intent, hospital based [48–53] Expensive PCR cDNA very low limits of detection procedure, commercial assay (4) Multiplex Simultaneous detection of multiple Research intent, hospital based [54–57] Use of multiple primer and/or probe sets Less sensitive PCR pathogenic species or strains procedure Very low limits of detection, improved A combination of immunoassay and Research intent, not (5) Immuno-PCR [58, 59] limits of detection over individual ELISA, Complex experimental design real-time PCR commercialized and PCR (4000 and 4 fold. respectively) Hybridization of sample biomolecules to Highly sensitive, large scale identification Research intent, hospital based (6) Microarray [60–74] immobilized target DNA or protein on a of multiple pathogens; protein and Cost-ineective procedure, commercial assay chip nucleic acid targets (D) Biophysical method Highly sensitive and specific even at Mass spectroscopy of PCR-amplicons Research intent, not (1) PCR-ESI-MS [75, 76] strain level and efficient multiple Expensive through electron spray dispersion commercialized pathogens detection. Inelastic scattering of monochromatic radiation upon interaction with an Rapid and nondestructive detection of Research intent, not (2) SERS [77–84] Sample preparation analyte with low-frequency vibrational analytes with high sensitivity commercialized and/or rotational energy Advances in Virology 5 within first 3 days aeft r onset of symptoms, but the sensitivity pathogens like RNA viruses, DNA virus, or bacteria [43]. decreases if tested 4–7 days aer ft the onset of the symptoms Although this method is rapid, the requirement of the [24]. DFA can detect RSV alone or as a multivalent test turbidimeter makes it slightly inconvenient as a cost-effective for other respiratory viruses (influenza A and B viruses, detection technique. Development in the chemistry of the parainfluenza virus types 1 to 3, and adenovirus) using LAMP has made it possible to get rid of the turbidimeter as SimulFluor Respiratory Screen assay [25]. it is now possible to visually detect the presence of pathogen in the test sample. LAMP is now referred to as colorimetric detection of loop-mediated isothermal amplification reaction 2.1.3. Optical Immunoassay. The direct visualization of an due to incorporation of the dye in the chemistry to aid visual antigen-antibody complex often referred to as optical immu- detection. Wang et al. (2012) have developed such an RT- noassay (OIA) is used for qualitative detection of Streptococ- LAMP assay for the detection of human metapneumoviruses cus and influenza virus. Visualization of immunocomplexes (which include rhinovirus, RSV, influenza virus A/PR/8/34 canbeenhancedbyimmobilizingthemonaspecialreflecting (H1N1)), which can detect as low as ten viral RNA copies surface. eTh antigen-antibody complex forms a thin layer, with better efficiency than RT-PCR [ 44]. It is now possible which changes the reflective properties of the surface [ 36]. to detect RSV A and B strains in nasopharyngeal specimen The technique is simple, rapid, and sensitive and as low as using multiplex LAMP (M-LAMP) in just 30 minutes. M- 1 ng of antibody per mL can also be detected [37]. OIA is LAMP had a sensitivity and specificity of 100% when com- now used for detection of RSV with a sensitivity, specicfi ity, pared with PCR [45]. positive, and negative predictive values of 87.9%, 99.6%, 98.9%, and 94.5%, respectively. Though OIA oer ff s rapid and cost-eeff ctive RSV detection, it is recommended that negative 2.3. Polymerase ChainReaction(PCR) BasedDetection results of OIA should be conrfi med by other tests [ 38]. 2.3.1. Conventional PCR. The applicability of PCR is ubiqui- tous andhas made thediagnosis of pathogenstremendously 2.1.4. LateralFlowImmunoassay. Lateral flow immunoassay rapid and sensitive [46]. RSV is usually challenging to detect (LFIA) is an immunochromatographic technique known for due to poor viral titer and sensitivity to antigen based detec- the rapid detection of RSV from nasal washes or nasal tion methods. u Th s, a PCR based method was developed and aspirates. The lateral flow of antigen-antibody complex on compared to serological and culture based detection methods the substrate matrix reaches the reaction area and results in in adults having respiratory infections [47]. The PCR method formation of colored band indicating the presence of antigen was based on the reverse transcription-nested PCR technique in thespecimen. eTh re aremanyLFIAkitslikeRemel Xpect, involving the outer and inner primers designed from the Binax Now RSV, BD Directigen EZ RSV, QuickLab RSV Test, F gene of RSV strain A, over a two day procedure with a and RSV Respi-Strip [39–42]. eTh sensitivity and specicfi ity sensitivity of 73%. This method is faster as compared to the are normally above 90% and 95%, respectively, but vary as conventional culture method which usually takes 3–5 days, per the manufacturer. Modifications of LFIA may include resulting in faster treatments. colloidal gold conjugated with antibody specific to RSV in thematrixfor trapping theantigen andassisting thegold mediated reaction for color band development. 2.3.2. Real-Time PCR. Although conventional reverse tran- scriptase-PCR (RT-PCR) is sensitive as compared to the 2.2. Loop-Mediated Isothermal Amplification. Loop-mediat- culture methods, it suffers from the lower sensitivity. This ed isothermal amplification (LAMP) is a nucleic acid based problem has been obviated by the development of real-time detection method used for bacterial and viral pathogens. It PCR based methods. Real-time PCR is by far the most can be used for RNA viruses where the additional step of sensitive method for the detection and diagnosis of a wide cDNA synthesis is required and commonly designated as array of pathogens, including RSV. Several studies have been reverse transcription LAMP (RT-LAMP). LAMP consists of conducted for the development of real-time PCR assays for pairs of primers specific to cDNA, which is then amplified by RSV detection, especially the RT-PCR. In one such study, autocycling strand displacement activity of DNA polymerase a rapid, sensitive, and specific method was devised based generally at 60 C. eTh reaction time of 1 to 1.5 hours is on TaqMan real-time PCR for the detection of both RSV A sucffi ient and can be monitored by real-time turbidimeter and RSV B strain [48]. The primer and the probe sets were and the resulting product can also be viewed by agarose designed from the nucleocapsid gene (N). The sensitivity of gel electrophoresis. This method can distinguish RSV strain this method was found to be 0.023 PFU (plaque forming unit) Aand Buponrestriction digestionofthe product[41]. or two copies of mRNA for RSV A, whereas the sensitivity Alternatively, RSV strain A and B can be detected designing for RSV B was 0.018 PFU or nine mRNA copies. This method specific primer sets for them. eTh efficiency of RT-LAMP is fast and efficient as the diagnosis was performed within 6 was tested for RSV detection from nasopharyngeal aspirates hours of the procurement of samples. and compared with viral isolation, enzyme immunoassay, A similar assay based on real-time PCR was developed immuno-chromatographic assay, and real-time PCR. RT- and employed in the detection of RSV in the bronchoalveolar LAMP was the most sensitive among all the methods tested, lavage (BAL)oflungtransplantpatientsorpatientswith with the exception of real-time PCR. Also, the RT-LAMP was respiratory infections [49]. The assay was designed based on specific for RSV and did not react with any other respiratory theRSV Ngeneand rfi st involved ascreening step of theRSV 6 Advances in Virology positive samples using a SYBR green based assay, followed 2.4. Microarray. Correlating gene expression signatures with by quantitative real-time PCR using TaqMan based assay to disease progression of the patient’s individual genetic profile be more cost eeff ctive. Also, the assay was developed for the is possible by comprehensive understanding and interpre- detection of both RSV A and B subgroups separately. The tation of the dynamics of biome-interactions in the lungs. assay was found positive in 16% of transplant patients, thus This would result in more efficient therapy for respira- indicating some possible association between RSV infection tory diseases via the concept of personalized medicine, and lung transplant tolerance. wherein the microarray finds its applications. Microarrays Real-timePCR basedonRSV Ngenehas also been used are the miniaturized assay platforms with a high-density to quantify RSV from the nasal aspirates of children [50]. array of immobilized DNA or protein. Hybridization of The method was one log more sensitive than the conventional sample biomolecules to corresponding DNA or protein on culture method. The real-time PCR assay resulted in 56% of the chip is detected, which allows determination of a variety positives, whereas the immunou fl orescence assay had 41.3% of analytes present in the samples in a single experiment and culture method had 45.3% of positives. Surprisingly, the [60]. Microarray has proved to be a robust and reliable RSV to GAPDH ratio did not differ in children with severe or tool to understand the echelon of genomics, transcriptomics, nonsevere infection, raising confusion about the correlation and proteomics. Implementation of microarray with the of viral load to the severity of infection due to the intensity metagenomic approach serves for rapid virus identifica- of viral replication, genetic susceptibility of the host and tion. Microarray based identification and characterization immune responses, thus, making it necessary to consider of viruses in clinical diagnostics are possible by designing other factors besides the viral load. oligonucleotide probes using the sequence data available in A similar assay was developed for the detection of RSV the public database. This approach is capable of discovering using primer-probe sets from the RSV F gene [51]. A com- novel viruses, even though there are no conserved genes that parative analysis was made between the nested RT-PCR, real- can be targeted by sequencing. In the case of respiratory time PCR, and ELISA, wherein real-time PCR was 25% more diseases, it can be executed by collecting the bronchoalveolar sensitive than the conventional nested PCR, thus making it lavage enriched by nuclease treatment followed by filtration more applicable for clinical samples. Additionally, the real- and extraction of total nucleic acids. Further, the nucleic acids time PCR was performed in two steps to increase the sensitiv- are amplified by a random priming-based method, referred to ity of the samples. This method was more rapid as the results as sequence independent single primer amplification, giving were obtained in 3.5 to 4 hours upon receiving the samples. near-full-length reads of genomes of RNA or DNA viruses, Several other reports also emphasize the applicability of real- which could then be compared to known viruses and used time PCR in detection of RSV in immunocompromised for designing the oligomers for the microarray [61]. u Th s, patients [52]. Besides the RSV F and N gene, the RSV matrix sequencing coupled with microarray is a powerful high gene andpolymerasegenehavealsobeenusedtodetectRSV throughput diagnostic tool. in children [53]. The latter method had the ability to quantify The utility of a metagenomics based strategy for broad- and classify RSV with high efficiency and rapidity. However, spectrum diagnostic assay using microarrays was demon- the sensitivity of the technique relies on the age of the strated for screening viruses like Rhino virus, Parainfluenza patients. virus, Sendai virus, Poliovirus, Adenovirus, and RSV from clinical samples [62]. A well-known example is “Virochip” which is a pan-viral microarray, designed to simultaneously detect all known viruses, and has comparable or superior sen- 2.3.3. Multiplex PCR. The multiplex PCR approach has been used for the detection and subtyping of RSV and human sitivity and specificity to conventional diagnostics [ 63, 64]. influenza virus simultaneously [ 54] and as many as 18 Similarly, an influenza microarray, “FluChip-55 microarray”, respiratory viruses could be detected [55]. The method was for the rapid identification of influenza A virus subtypes based on the primer sets of hemagglutinin and nucleoprotein H1N1, H3N2, and H5N1 was developed [65]. The procedure is simple and follows few steps including RNA extraction from gene of influenza virus and nucleocapsid protein gene of RSV and found to be rapid, specific, and sensitive. However, clinical samples, subsequently their reverse transcription, the method suffered from the drawback that it could not second-strand cDNA synthesis, and then PCR amplification of randomly primed cDNA. eTh hybridization of nucleic distinguish between RSV A and B. This method could prove significant during a respiratory outbreak for surveillance. In acidswithprobesisdonebyfluorescentdyesorbyan such situations, multiplex real-time PCR assay, such as the alternative method of electrochemical detection. eTh latter commercial “Simplexa Flu A/B & RSV kit” that differentiates method relies on a redox reaction to generate electrical influenza A virus, influenza B virus, and RSV, is very useful current on the array for measurement. Incorporation of Cy3 [56]. Likewise, recently, a rapid and sensitive detection assay u fl orescent dye and hybridization to the microarray [ 66] for RSV and other respiratory viruses was developed by has been empowered by specific algorithms to match the Idaho Technologies christened as FilmArray (Idaho Tech- diagnostic needs, leading to the final and critical step of scanning and analysis [67]. nologies, Salt Lake City, UT). This is an automatic real-time molecular station with capability of nucleic acid extraction, The genes involved in the pathways of neuroactive ligand- initial reverse transcription, and multiplex PCR followed receptor interaction, p53 signaling, ubiquitin mediated prote- olysis, Jak-STAT signaling, cytokine-cytokine receptor inter- by singleplex second-stage PCR reactions for specific virus detection [57]. action, hematopoietic cell lineage, cell cycle, apoptosis, and Advances in Virology 7 cancer were upregulated in RSV-infected BEAS-2B cells. RSV mass spectrometry (Py/GC/MS), Capillary electrophoresis- infection up-regulated 947 and 3047 genes at 4 h and 24 h, MS, and Liquid chromatography mass spectrometry (LC- respectively, and 124 genes were common at both instances. MS) are the few examples that are used for the identification Moreover, 1682 and 3771 genes were downregulated at 4 h and of pathogens [89]. But the MS technique that promises 24 h, respectively, and only 192 genes were same. Respiratory robust practical application for pathogen detection is the disease biomarkers like ARG2, SCNN1G, EPB41L4B, CSF1, electrospray ionization mass spectrometry (ESI-MS) [92, 93] PTEN, TUBB1, and ESR2 were also detected. RSV infection Figure 3(a). signs and symptoms render a partial consequence of host A notable ESI-MS for global surveillance of influenza pathogen interaction, but the transcription profile is better virus was accomplished by Sampath et al. [75]bycoupling exemplified by microarray. eTh transcription profiles of RSV MS with reverse-transcription PCR (RT-PCR). eTh study cor- infected mice lungs and lymph nodes showed gene expression rectly identiefi d 92 mammalian and avian influenza isolates of antigen processing and inflammation. The response is (which represented 30 different H and N types, including 29 higher in lungs on the day 1 aer ft RSV infection than day 3. avian H5N1 isolates). eTh analysis showed more than 97% The gene expression profile shortly aer ft RSV infection can be sensitivity and specificity in the identification of 656 clinical accountedasabiomarkerandcanbescaledupfordiagnostics human respiratory specimens collected over a seven-year with in vitro and in vivo profiles [ 68, 69]. period (1999–2006) at species and subtypes level. The surveil- Sometimes DNA microarrays do not confer their utility lance of samples from 2005-2006 influenza virus incidence in very specicfi investigations in the case of personalized showed evidence of new genotypes of the H3N2 strains. eTh medicine wherein the manifestation of disease occurs at the study also suggested approximately 1% mixed viral quasi- transcription level. This situation may arise in complications species in the 2005-2006 samples providing insight into viral of RSV with other associated disorders like asthma [70, evolution. This study led to a number of RT-PCR/ESI-MS 71]. As protein is the abundant functional biomolecule, based detection methods for RSV and related respiratory reflecting the physiological or pathological state of the organ, viruses like influenza A and B, parainfluenza types 1–4, protein microarray profile is an option to access under adenoviridae types A–F, coronaviridae, human bocavirus, and this situation [72]. Protein microarray has analytical and human metapneumovirus screening [76]. These assays had functional applications to study the protein-protein, protein- 87.9% accuracy, compared to conventional clinical virology DNA, and protein-ligand interactions. es Th e features enable assays and pathogens undetected by traditional clinical virol- the profiling of immune responses and are thus important ogy methods could be successfully detected. The advantage for diagnostics and biomarker discovery. Protein microarray of RT-PCR/ESI-MS platform is the ability to determine the can serve as a rapid, sensitive, and simple tool for large-scale quantity of pathogens, detailed pathogen characterization, identification of viral-specific antibodies in sera [ 73, 74]. An and the detection of multiple pathogens with high sensitivity extensive study was done during the 2002 SARS pandemic and ecffi iency. using coronavirus protein microarray to screen antibodies in human sera (>600 samples) with>90% accuracy and at least 2.6. Nanotechnology Based Detection. Recent advancements as sensitive as, and more specific than the available ELISA in nanotechnology have changed the perception and perspec- tests [73]. u Th s, this system has enormous potential to be used tive of research.Whenscaleddowntonanometers, theprop- as an epidemiological tool to screen viral infections. erties of matter change and nanotechnology exploits these new properties and harnesses them with the existing tech- 2.5. Mass Spectroscopy. Mass spectroscopy (MS) has become nologies to exceptional capabilities. Techniques discussed a method of choice for molecular investigation of pathogens below are examples of the nanotechnology based detection as the reliability is reinforced due the well-characterized approaches which utilize basic traditional but indispensable sequence information of nucleic acids or proteins and even detection techniques. for intact viruses [86, 87]. But pragmatic usage of MS is possible when coupled with various chromatography and 2.6.1. Nanoparticle Amplified Immuno-PCR. Immuno-PCR, affinity-based techniques. eTh combination of affinity based a combination of ELISA and PCR, is used widely for the viral detention and nucleic acid based MS serves as a solution detection of various bacterial and viral antigens with lower for low detection limits. Affinity-based methods employing titers as meagre as zepto moles [58]. Perez et al. [59]reported nanotechnology can be used to nd fi traces of target pathogen a modification of immuno-PCR using gold nanoparticles to improve detection limits. PCR amplification of pathogen for RSV detection. Target extraction was enhanced by using nucleic acid combined with MS can be used as substitutes magnetic microparticles (MMPs) functionalized with anti- [88–90]. MS has the advantage of rapid identification of mul- RSV antibody to capture the antigen (RSV). The MMP- tiple viruses at the same time and even identifies the protein RSV complex is then countered with gold nanoparticles modification status [ 86, 91]. Now, MS is no longer confined functionalized with both, palivizumab (Synagis), an anti-RSV to proteomics based analysis, and MS based genomics have F protein antibody, and DNA sequence partially hybridized become common practice. er Th e are several variations of with a tag DNA sequence (fAuNP). eTh MMP-RSV-fAuNP MS, like Matrix-assisted laser desorption/ionization mass complexisthenheatedtorelease thepartially hybridized spectroscopy (MALDI-MS), Surface-enhanced laser desorp- tag DNA sequence, which is then quantiefi d from the tion/ionization mass spectroscopy (SELDI-MS), Bioaerosol supernatant by real-time PCR. These modifications enabled mass spectrometry (BAMS), Pyrolysis gas chromatography detection of RSV even at 4.1 PFU/mL. This assay oer ff s 8 Advances in Virology PCR amplicon ESI-MS Identification of virus Virus A Virus B Virus C Virus D (a) PCR-Electrospray Ionization/Mass Spectroscopy Silver film Silver nanorods Virus A Virus B Virus C Virus D Laser Spectra Silver vapor source Silver nanorods Oblique vapor desposition (b) Surface Enhanced Raman Spectroscopy Figure 3: A schematic representation of biophysical method of RSV detection. (a) PCR-electrospray ionization mass spectroscopy and (b) Surface enhanced Raman spectroscopy. a 4000-fold improvement in the limit of detection over ELISA possible using DNA hairpin structures functionalized to and a 4-fold improvement over detection when compared a gold filament, which is immersed in a capillary tube with real-time RT-PCR [59]. containing viral RNA and scanned for u fl orescence. eTh set- up wasabletodetectaslow as 11.9PFUs, whichwas∼200 2.6.2. Live Cell RNA Imaging. Live cell imaging can pro- times better than a standard comparison ELISA. vide the benefit of eeff ctive diagnosis and treatment, if the capability of identifying, monitoring, and quantifying 2.6.3. Quantum Dots. Immunou fl orescence microscopy biomolecules is developed. However, development of such based detection of RSV, that is, direct uo fl rescent-antibody systems for detection of viral agents is dicffi ult, especially in assay (DFA), is considered as gold standard [39], but the the early stages of infection. With the advent of molecular comparativeecffi acyofDFA with otherassaysdoesnot seem beacon technology, it is now possible to track mRNA of to have reached a consensus [39, 40, 94–97]. The prime host and RSV [32]. Based on this framework, a modifica- possible disadvantages and inconsistencies of DFA can be tion by oligonucleotide-functionalized gold nanoparticulate attributed to the fading of the dyes, conjugating antibodies probe was suggested as an improvement, wherein a gold with dyes, limited sensitivity due to background staining, and nanoparticle is functionalized with DNA hairpin structure by excitation at two different wavelengths [ 26–28]. To address thiol linkage. eTh DNA is so designed that the loop portion these issues, uo fl rescent nanoparticles, that is, quantum has a complimentary sequence to RNA to be detected and dots (QDs), appear as promising candidates for field clinical the 5 stem is linked to gold nanoparticle by thiol group and diagnostics. Due to their inorganic nature, they are less the 3 endislinkedtoafluorophore.Onhybridization to the susceptible to metabolic degradation. QDs are photostable; target RNA with loop, the uo fl rophore goes away from the that is, they do not lose u fl orescence on long exposure to light quenching gold and the emission is tracked. us, Th live imag- and can be excited at the same wavelength while radiating at ing of mRNA is possible. This mechanism of hairpin DNA different wavelengths and hence can be used for multiplexing functionalized gold nanoparticles (hAuNP) was executed by [27]. Various successful attempts were made to ameliorate Jayagopal et al. in detecting RSV mRNA in HEp-2 cells [33]. RSV detection using QDs. eTh progression of RSV infection This technique oer ff s the advantage of real-time detection in the HEp-2 cell line was studied using confocal microscopy of multiple mRNA at the same time, including the mRNA by QDs probing F and G proteins and it was found that of RSV and glyceraldehyde 3-phosphate dehydrogenase of this method was more sensitive than real-time, quantitative the HEp-2 cell. Quantitative assay using this approach was RT-PCR, particularly at early infection [29]. This approach Advances in Virology 9 was used by Tripp et al. in vitro,onVerocelllines and spectral signature [81]. Bacteria and viruses from various was extrapolated by an in vivo BALB/c mice study, which biological samples can be identiefi d, characterized, and clas- concluded the approach beyond diagnostics as it can be used sified from clinical samples [ 77, 82]. SERS can distinguish for multiplexed virus and/or host cell antigen detection and between DNA or RNA viruses like Adenovirus, Rhinovirus, intracellular tracking studies [28]. Rotavirus [83], and RSV [84]. Rotavirus is the common cause Flow cytometry is now widely used in diagnostics and the of gastroenteritis in children and the rapid and sensitive reliability is improved because millions of cells are analyzed detection was demonstrated with SERS [83]. SERS is an at a time and comprehensive data is produced [30]. Tracking established powerful tool for sensitive, expedited, and specific and targeting cellular proteins and the ease of multiple detection of various respiratory pathogens, like Mycoplasma parameters correlation allow flow cytometry to be used for pneumoniae and RSV. Using silver nanorod arrays (NA) various qualitative and quantitative assays. Flow cytometer platform for SERS, differentiation of M. pneumoniae in could detect RSV with sensitivity and reproducibility [31]. cultureand in spiked andtrueclinicalthroatswabsamples Agrawal et al. [26] showed that antibody conjugated QDs was achieved [82]. The notable sensitivity of SERS to resolve could detect RSV rapidly and sensitively using the principles strain level differences for RSV strains A/Long, A2, ΔG, and of microcapillary flow cytometry (integrated with a fixed- B1 can be exploited for clinical diagnosis instrumentation point confocal microscope) and single-molecule detection. [84]. A 40-nm carboxylate-modified uo fl rescent nanoparticles and streptavidin-coated QDs were used in their study, which 3. Prevention could estimate relative levels of surface protein expression. Prevention is the most important aspect of healthcare and 2.6.4. Gold Nanoparticle Facilitated Microarray. The FDA has prime contribution to the culmination of the disease approved microarray systems, semiautomated respiratory than the treatment measures. Effective preventive measures virus nucleic acid test (VRNAT), and the fully automated res- reduce the mortality and economic burden of the disease. piratory virus nucleic acid test SP (RVNATSP)(Nanosphere, Untilnow thereisnoeeff ctivevaccine forpreventionfor RSV. Northbrook, IL) are examples of the microarray based detec- Direct or indirect contact with the nasopharyngeal secretions tion systems for influenza A virus, influenza B virus, RSV or droplets (sneezing, coughing and kissing), fomites, and A, and RSV B [56, 98]. eTh se systems are based on the e-ffi food from infected patients can potentially transmit RSV. Live cient detection of microarray based hybridization using gold viruscansurviveonsurfacesforseveralhours[100],butatthe nanoparticles. The hybridization between the oligonucleotide cellular level, the viral spread is a series of systematic events probes and target DNA/RNA is detected specifically by of invasion including viral attachment and fusion followed hybridizing them again to gold nanoparticles functionalized by viral replication and protein synthesis. The F protein with oligonucleotide and the signal is generated by gold accumulates in the host membrane and then surrounds the facilitated reduction of silver in the presence of a reducing budding progeny viruses, thus spreading the infection to agent [99]. adjacent cells and exacerbating the infection (Figure 4). u Th s these proteins are considered as the potential candidate for 2.6.5. Surface Enhanced Raman Spectroscopy. Raman spec- the development of prevention measures such as antibodies, troscopy works on the basis of the inelastic scattering of DNA vaccines, and subunit vaccines. monochromatic radiation like near infrared, visible, or near ultraviolet, interacting with an analyte with low-frequency vibrational and/or rotational energy. But the signal generated 3.1. Antibodies. RSVhas threeenvelopeproteinsF,G,and is low and hence the signal is enhanced using silver or gold SH. Both F and G are glycosylated and represent the targets matrix substrates. er Th e are many modifications of Raman of neutralizing antibodies. eTh RSV F protein emerged as spectroscopy,but themostwidelyusedone is thesurface a good vaccine candidate due to its conserved and vital enhanced Raman spectroscopy (SERS) (Figure 3(b))[77]. role in cell attachment. Passive immunization is a direct Direct intrinsic, indirect intrinsic and extrinsic detection are approach to counter RSV (Figure 5). Initially, polyclonal three major SERS detection congfi urations [ 78]. In contrast antibodies from healthy human individuals resistant to RSV to IR spectroscopy,the Ramanspectra canbeobtained were successful in preventing RSV infection in high risk without the interference of water molecules and thus bio- infants and these pooled and purified immunoglobins were logical analytes can be studied in their native conformation. popular as RespiGam. The monoclonal antibody specifically SERS is routinely used for various bioanalytical purposes neutralizing F protein conferred eeff ctive protection against due to the rapid and nondestructive detection of analytes RSV as compared to RespiGam, and this licensed monoclonal with sensitivity, specificity, and precision even for a single antibody, palivizumab (Synagis), is now used to passively molecule or live cells [79, 80]. SERS can be targeted to protect high risk infants from severe disease, thus replacing various analytes that may constitute DNA, RNA, proteins, the RespiGam. The efficacy of the recombinant monoclonal or other organic compounds. Nucleic acids are the preferred antibody, palivizumab, has been tested for prophylaxis and candidates in biological SERS investigations, as the influence therapy in immunocompromised cotton rats [101]. Repeated of base composition and sequence, conformation (local doses of palivizumab were required to prevent rebound and/or global) of nucleic acids, or intermolecular dynamics RSV replication. Palivizumab is administered alone or in with protein or ligand is correspondingly expressed as typical combination with aerosolized ribavirin. Palivizumab cannot 10 Advances in Virology RSV Life cycle of RSV Budding RSV ready to infect adjacent cell Merging with the cell membrane Antigenome F protein Transcription Replication G protein Protein synthesis Assembly SH protein P protein N protein L protein Golgi complex Endoplasmic Nucleus reticulum Figure 4: A schematic representation of RSV life cycle. Modes of RSV inhibition G protein F protein + + No infection No binding to the cell RSV Gold nanoparticles Cell No infection + + No binding to the cell RSV Antibodies Drug No infection No binding Peptide to the cell RSV Fusion inhibitors Infection Fusion No treatment Binding RSV Figure 5: A schematic representation of various compounds inhibiting RSV binding to the cell. cure or treat serious RSV disease but neutralization of RSV palivizumab or motavizumab when pretreated with RSV, but can help in preventing serious RSV infections. Motavizumab there was a reduction in viral transcription, thus inhibiting has been found to neutralize RSV by binding the RSV both cell-cell and virus-cell fusion most likely by preventing fusion protein F aer ft attachment to the host, but before the the conformational changes in the F protein needed for viral viral transcription [102]. Viral entry was not inhibited by fusion. Advances in Virology 11 The effective use of palivizumab is limited due to the cost of RSV for IFN and immunopathology, a construct was and its use in infants with high risk of bronchiolitis based on designed by inserting the F gene into Newcastle disease the coverage by different healthcare systems [ 103]. In spite virus (NDV) vector (NDV-F). This modification served the of these restrictions on palivizumab, it has a wide societal purposeofhigherelicitation of IFNbyNDV-F than RSV, impact on use in infants with chronic lung disease due to protection against RSV infection without immunopathology premature birth or those with haemodynamically signicfi ant and enhanced adaptive immunity in BALB/c mice [109]. Wu cardiac disease. According to the modified recommendations et al. [110] developed a DNA vaccination strategy against of the Committee on Infectious Diseases of the Centers for RSV using a mucosal adjuvant. Two DNA vaccine vectors, Disease Control and Prevention of RSV, palivizumab is rec- namely, DRF-412 and DRF-412-P containing residues 412– ommended for infants with congenital heart disease (CHD), 524 of the RSV F gene, were cloned into the phCMV1 chronic lung disease (CLD), and birth before 32 weeks [104]. DNA vaccine vector. eTh DNA vaccine vectors DRF-412 Minimum 5 doses are recommended irrespective of the contained the cholera toxin gene region called ctxA2B acting month of the first dose for all geographical locations for as a mucosal adjuvant. eTh DNA vaccine was successfully infants with a gestational age of 32 weeks 0 days to 32 weeks expressed in mouse muscle tissue, which was conrfi med by 6 days without hemodynamically significant CHD or CLD. immunohistological analysis and RT-PCR. The immunized The new recommendations of the committee were aimed at mice induced neutralization antibody, systemic Ab (IgG, the high risk groups including infants attending child care IgG1, IgG2a, and IgG2b) responses, and mucosal antibody or one or more siblings or other children younger than 5 responses (Ig A) which mimicked the challenge with live RSV. years living with the child. Also, the infants were qualified The mice immunized with the DRF-412 vector contained less for receiving prophylaxis only until they reached 90 days of RSV RNA in lung tissue and induced a higher mixed Th1/Th2 age. Palivizumab, although eecti ff ve, is costly and thus is not cytokine immune response and had better protection than benecfi ial to the recipients especially during the periods when those immunized with the DRF-412-P vector, which was RSV is not circulating. A cost eeff ctive means of producing confirmed by lung immunohistology studies [ 110]. A rational RSV F neutralizing antibodies was experimented in phages approach to confer protection against various pathogens and plants. Much success in this regard of palivizumab through single vaccination or more often known as combined production was observed in the Nicotiana benthamiana plant or composite vaccines was employed for RSV, influenza A system which oeff red glycosylation and high production virus (INF-A), and herpes simplex virus type-1 [111]. Mice at lower upstream and equivalent downstream cost, when were immunized either by injection or by gene gun (gold compared to mammalian derived palivizumab. The efficacy of beads as carrier of the plasmid DNA) with mixture of four the plant derived palivizumab was more than the mammalian plasmids: INF-A haemagglutinin (HA), INF-A nucleoprotein derived palivizumab or the plant derived human monoclonal (NP), HSV-1 glycoprotein D (gD) and RSV glycoprotein F. antibodies in cotton rats [105]. This led to protection of mice from the respective pathogens of challenge; in addition, it also offered protection from 3.2. DNA Vaccines. RSV genome codes structural and func- Mycoplasma pulmonis challenge as well [111]. Recently several tional proteins that are immunogenic and referred for vaccine developments have been made in the eld fi of recombinant vaccines. In one such approach, Mycobacterium bovis Bacillus development against RSV. DNA based vaccines were devel- oped basedonthese proteins becausethe conceptwas simple, Calmette-Gue’rin (BCG) vaccine was modified to carry RSV as it involved a DNA fragment coding part or whole protein of NorM2and wastestedand foundtoestablish theTh1type immunity in RSV challenged mice [112]. The recombinant RSV that was inserted into an appropriate expression plasmid vector under a constitutive promoter control (Figure 6). vaccine also elicited the activation of RSV specific T cells eTh initial work with this approach was successful in the producing IFN-𝛾 andIL-2, alongwithreduction in weight expressionincells and in vivo murine models to eliminate loss, and lung viral protein load, thus establishing a Th1- the RSV infection, but the problem of RSV associated Th2 polarized immune response. Bactofection is bacteria mediated transfer of plasmid type immune response was persistent. This problem was attempted to be resolved by manipulating the parameters DNA into mammalian cells. Xie et al. employed this interest- of choice of the protein to be expressed, the expression ing way of delivering and expressing the DNA vaccine in mice against RSV [113]. This approach serves the purpose of natu- vector, adjuvants, formulations, and intracellular stability of the plasmid. rally activating the immunostimulatory response of the host Mice challenged with the RSV-G construct had balanced andalsodeliveryofthe DNAvaccine.Attenuated Salmonella systemic and pulmonary Th1/Th2 cytokines and RSV neutral- typhimurium strain SL7207 expressing vector pcDNA3.1/F izing antibody responses [106]. However, the RSV F protein containing the RSV F gene was orally administered to BALB/c gene is considered as a widely used and prospective target mice, triggering efficient antigen-specific humoral, cellular, for the development of vaccines and is oen ft a favourite and mucosal immunity [113, 114]. model for DNA vaccines against RSV [107]. But the wild A novel method of developing RSV DNA vaccine was devised by replacing the structural genes with RSV genes type RSV F protein expressed from DNA plasmid was poorly expressed, so a codon optimized DNA vaccine was in an attenuated strain of Venezuelan equine encephalitis designed for better in vivo expression and hence was more virus (VEEV). VEEV has an ssRNA (+) genome and con- tains a strong subgenomic promoter. eTh replicon particles immunogenic and reduced the RSV titer [108]. To address the low immunogenicity of F protein, the antagonistic activity were prepared by providing helper RNAs encoding VEEV 12 Advances in Virology DNA functionalized DNA encapsulated DNA vaccine nanoparticles nanoparticles Viral DNA Naked DNA vector Expression vector RSV Vector Mice protected Vaccination RSV challenge RSV No vaccination RSV challenge RSV infection Figure 6: A schematic representation of a simple DNA vaccine administered as a naked DNA vector or functionalized nanoparticle or as an encapsulation for controlled delivery. capsid andenvelopeglycoproteinwhich comprise structural virus like particles (VLP) developed from NP and M proteins proteins, and all of these, when transfected into Vero cells, of Newcastle disease virus (NDV) and a chimeric protein resulted into a replicon particle that could independently consisting of cytoplasmic and transmembrane domains of synthesize the RSV protein, thereby activating the immune NDV HN protein and ectodomain of the RSV G protein response and protection. eTh system could be modulated by (H/G) [122]. These VLPs resulted in an immune response the administration of helper RNAs. This strategy was applied better than UV-inactivated RSV and provided complete to the mice and rhesus monkey models, conferring protection protection in mice from RSV even at a single dose, with against RSV and a desirable extent of a balanced Th1/Th2 type elicited neutralizing antibodies. eTh VLP-H/G-immunized immune response was received [115]. mice did not show enhanced pathology as compared to FI-RSV.Inanother approach to solvethe immunogenicity relatedproblemsindevisinganRSV vaccine,arecombinant 3.3. Subunit Vaccines. Several approaches have been con- sidered for developing an eeff ctive vaccine against RSV vector, based on either murine PIV type 1 or Sendai virus was used to deliver RSV G protein through reverse genetics [123]. including the formalin inactivated RSV vaccine developed in This provided effective protection against RSV in cotton late 1960s, which instead resulted in an enhanced infection [116]. An efficient RSV vaccine would be the one with proper rats. Another similar study using Sendai virus (SeV) as a vaccine carrying RSV F gene provided protection against four balance between immunogenicity and protection without any pathogens including hPIV-1, mouse PIV-1, hPIV-3, and RSV allergic response [8]. RSV F has widely been accepted as the vaccine candidate [124]. This approach has been used to compare two vaccine models, one with SeV backbone and the other with PIV-3 due to its conserved nature among various strains as well as among the other paramyxoviruses [8, 117–119]. An antigenic backbone. eTh SeV based vaccine showed a decrease in RSV load in African green monkeys lung titers. region corresponding to RSV F protein (region 255–278) Another study with recombinant vaccine utilizes recom- was cloned into a vector with ctxA2 B gene of cholera toxin and named as rF255, which elicited a helper T cell binant simian Varicella viruses (rSVVs) which express RSV G and M2 protein genes and was evaluated in the Vero type 1 immune response in mice. It also resulted in higher cell line [125]. Such a recombinant vaccine approach could expression of serum neutralizing antibody in immunized mice [120]. Similarly, a multivalent recombinant protein was be very useful in elderly people with risk of infection with Varicella andRSV,aswellaschildrenwithchickenpoxand developed by cloning RSV F, M2, and G protein into a bacterial pET32a (+) vector (called rFM2G), which resulted RSV. rSVV based vaccines resulted in enhanced immune responses to RSV antigens serving as suitable vaccines in in enhanced serum IgG titers [121]. rFM2G was also used rhesus monkeys. A very similar approach was adopted for in conjunctionwithflagellinasanadjuvantwhich didnot increase the IgG titers. In an eo ff rt to address the issues of developing a recombinant alphavirus or immune stimu- lating complex (ISCOM) antigen against RSV [126]. The immunogenicity, several studies have used the approach of recombinant vector was designed by using a self-abortive combining other viruses with RSV genes. One such study uses Advances in Virology 13 alphavirus called Semiliki forest virus carrying RSV F and and found to provide significant protection against RSV, G genes. eTh advantages of using such recombinant abortive along with RSV specicfi humoral responses and an enhanced viruses are that they can undergo viral infection just once, Th1/Th2 response [ 131]. The NE rendered the RSV ineffective eliminating chances of adverse eeff cts resulting from further when treatedfor2or3hours, with areduction in viraltiter infection, and infecting the host cell resulting in expression up to 10-fold as compared to media controls. NE-RSV also led of the inserted RSV gene followed by humoral and cell to higher expression of RSV specific antibody, with significant mediated immune response. eTh ISCOM used was composed decrease in viral load, no hyperreactivity, and no Th2 cytokine of Quillaja saponin, lipids, and RSV antigens, having adjuvant induction. u Th s, this novel approach seems to be a promising properties as well. ISCOM has been shown to enhance and safe vaccine option against RSV. antibody production, T cell proliferation, and MHC class- The bitter episode of formalin inactivated RSV vaccine I responses as well as RSV specific neutralizing antibodies, has impeded the vaccine development and in fact has raised IgG and IgA. The recombinant vaccine SFV/F or SFV/FG serious concern in the use of native RSV or its components. resulted in IFN gamma response along with resistance to This approach was revived with a novelty, wherein the RSV infection without worsened RSV disease, whereas the engineered RSV F protein aggregates formed nanoparticles resultswereoppositeinISCOM/FGwithenhancedgobletcell and were used as vaccine, and these nanoparticles induced hyperplasia post-RSV challenge. protective immunity in cotton rats [132]. To combat RSV, host neutralizing antibodies are always more preferred than the 3.4. Nanovaccines. Recently, several applications of nan- therapeutic antibodies. But the epitopes of the neutralizing otechnologyhaveappearedinthe developmentofvaccines antibodies are larger than those of the therapeutic neu- tralizing antibodies (palivizumab and motavizumab). us, Th popularly known as Nanovaccines. DNA vaccine is prone to rapid degradation when introduced into an animal system; the use of these epitopes for neutralizing antibodies as a so to increase the retention and increase the efficacy of the vaccine requires the retention of immuno functional con- DNA vaccines, they can be encapsulated into a polymer formation while getting rid of the undesired protein. RSV F that will protect and facilitate controlled release (Figure 5). oligomeric protein nanoparticle was synthesized by inserting recombinant RSV F gene into Baculovirus and expressed Various synthetic or natural polymers are now experimented for targeted delivery and controlled release of the carrier. in Sf9 insect cell lines. This resulted in high recombinant Chitosan is the polymer of great interest in respiratory protein expression compared to native protein. This also resulted in the conformation of rosette nanoparticles which disease treatment, because of its mucoadhesive property and biodegradability, which balances the purpose of longer reten- wasthe aggregateofmultipleRSV Foligomers foundtobe tion and controlled release of carrier molecules encapsulated. immunogenic. The study was extrapolated to phase 1 human u Th s, chitosan nanoparticles are being developed against clinical trials, which showed its safety and efficacy against RSV. A DNA vaccine (DR-FM2G) constitutive expression RSV [133]. vector consisting of antigenic regions of RSV F, M2, and G genes driven by human cytomegalovirus promoter was 3.5. Nutrition. Nutrition is not considered as treatment encapsulated using chitosan nanoparticles (DCNPs). The but is a prerequisite for homeostasis, and any nutritional advantages of DCNPs are that it was more stable than naked imbalance attracts disorders and diseases. Supplementary DNA or chitosan and so ideal for protection against DNA nutrients could reverse the adverse eeff cts of the disorders degradation by nucleases. The sustainability of the DCNPs in and diseases, so in sensu nutrition can serve both as treatment mice was more than naked DNA which was correspondingly and prevention. Carbohydrate rich diets and diet lacking indicated by higher RSV protein expression evident by antioxidants like fruits and vegetables during pregnancy are immunohistochemical and real-time PCR studies [127, 128]. suspected for RSV susceptibility [134]. Resveratrol (trans- A similar study was conducted by Eroglu et al. [129], where 3,4,5-trihydroxystilbene), a polyphenol from grapes has thehighlyconserved RSVFgene wasclonedintopHCMV1 demonstrated reduction in RSV replication and inflamma- expression vector and encapsulated into poly-hydroxyethyl- tion [135, 136]. A study demonstrated that the cord blood methacrylate nanospheres coated by chitosan and transfected deficient in vitamin D was associated with RSV bronchiolitis; into Cos-7 cells. eTh transfection efficiency of the system the neonates were at higher risk of RSV in the rfi st year was at par with commercially available transfecting agents. In of life [137]. Vitamin D inhibits NF-𝜅 B signalling which vivo studies with the BALB/C mice also indicated F protein is responsible for RSV inflammation without affecting the expression and reduced RSV infection. antiviralactivityofthe host [138]. Lower levels of micronutri- Avaccine basedonthe recombinantRSV Ngenerings, ents like zinc, copper, selenium and retinol (Vitamin A), and called N-SRS, enclosing a bacterial RNA has been developed alpha-tocopherol (Vitamin E) were observed in the children and assessed intranasally in BALB/c mice [130]. N-SRS was aeff cted by RSV and human metapneumovirus [ 139]. In one adjuvanted with E. coli enterotoxin LT (R192G) with efficient study, RSV infected children were administered vitamin A protection against RSV and high titers of IgG1, IgG2a and to compensate the lower vitamin A serum level which was IgA, andsoforth.Althoughthisnanovaccine elicited amild found benecfi ial for children with severe RSV infection [ 140]. inflammatory response in airways of the mice, it enhanced Also, probiotic diet has shown to boost resistance against + + the expression of antigen specific CD4 and CD8 responses. pathogen by modulating immune response, as in case of In another novel approach, nanoemulsion (NE) was used RSV, Lactobacillus rhamnosus (a probiotic bacteria) treated as an adjuvant to mucosal RSV vaccine in a mouse model BALB/c mice showed significantly reduced lung viral loads 14 Advances in Virology and pathology after the RSV challenge [ 141]. These studies shock protein (HSP90) for its replication [165]. The anti- indicate the importance and association of nutrition with RSV activity of these compounds was seen in human airway RSV susceptibility. epithelial cells (HAEC) and is considered as drug resistant therapeutics, due to the highly conserved target chaperon protein. eTh se compounds are also known to inhibit repli- 4. Treatment cation of HPIV, influenza virus, and rhinovirus. eTh anti- There are very limited treatment options available for RSV. viral activity dose is not toxic to the cells and inhalation However, there are many drugs for the symptoms associated mode of treatment can increase the local efficacy and avoid with RSV infection. eTh target genes and proteins vital for unnecessary exposure to other organs [166]. RSV infection (discussed in Section 3)are importantfor developing preventative and treatment measures. eTh mode 4.2. Fusion Inhibitors. Recent advances in the development of action and potency of a drug determines the approach of anti-viral drugs include the fusion inhibitors. eTh fusion of prophylactic or curative application. Considering the inhibitors are usually synthetic compounds or molecules proposed life cycle of RSV, theoretically, there are numerous interrupting the fusion of virus with the host cell usually by modes to interfere with RSV infection, but these options may binding the fusion proteins (Figure 5). The fusion inhibitors not be practical. Replication, transcription and fusion are the have been widely studied as anti-viral agents in several viruses few target processes for drug development against RSV. A including HIV, RSV, Henipavirus, Hendra virus, Nipah virus, focus is therefore on development of potent drug which holds Paramyxovirus, metapneumoviruses, HIV, and RSV [11, 167– conformity in the human trials. Some of the approaches are 173]. The first reports of the use of peptide(s) as fusion described below that promise to be a potential treatment for inhibitors include the development of DP-178, a synthetic RSV (Table 2). peptide based on the leucine zipper region of the HIV fusion glycoprotein gp41 [167], which showed an IC50 at 0.38 nM 4.1. Antiviral Drugs. Ribavirin or 1-[(2R, 3R, 4S, 5R)-3,4- against HIV-1. Fusion inhibitors for the paramyxoviruses dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-tri- have also been developed based on the conserved region azole-3-carboxamide is a widely used broad spectrum of the fusion protein F. The F protein is widely known for synthetic anti-viral drug for both DNA and RNA virus its conserved nature among the Paramyxoviridae family [7]. treatment. Oral and nasal administration of ribavirin Lambert et al. [174] developed the fusion inhibitors belonging for treatment of severe lower respiratory tract infections to the conserved heptad repeat (HR) domains of F1 region caused by RSV and influenza virus are an option [ 104]. of F protein which is analogous to the peptides DP-107 and Ribavirin is phosphorylated in the cells and competes with DP-178 of HIV gp41. These fusion inhibitors were tested 󸀠 󸀠 adenosine-5 -triphosphate and guanosine-5 -triphosphate against RSV, human parainfluenza virus 3, and measles virus, for viral RNA-dependent RNA polymerases in RNA which showed antiviral activity specific to the species of viruses. However, the mechanism of ribavirin differs for origin. DP-178 is an FDA approved anti-HIV drug with DNA viruses, as it is a competitive inhibitor of inosine International Nonproprietary Name (INN) Enfuvirtide and monophosphate dehydrogenase (IMPDH), causing deletion trade name Fuzeon. Out of the peptides tested, the peptide of GTP and messenger RNA (mRNA) guanylyl transferase T-118 developed from RSV was the most eeff ctive, with an (mRNA capping enzyme) and adversely affecting protein EC50 of 0.050𝜇 M. These fusion inhibitors were then further synthesis [142, 143]. The exact mechanism of anti-viral characterized and tested. It was shown that dieff rent fusion activity of ribavirin against RNA and DNA viruses is still inhibitors derived from same HR region differ in their anti- not clear. eTh usefulness of ribavirin against viruses is viral activity [147]. The HR121 and HR212 peptides showed an not only due to its anti-viral activity but also due to its IC50 of 3.3 and 7.95𝜇 M, respectively, against RSV. Similarly, capability to modulate the immune system. Ribavirin is another peptide inhibitor was developed from Rho-A which suggested to have immuno-stimulatory eeff cts on Th cells showed inhibition of syncytia formation induced by RSV [144]. The derivatives of ribavirin such as viramidine, [148]. RhoA (a small GTPase) is involved in many biological merimepodib, and other IMPDH inhibitory molecules processes and was shown to bind the RSV-F protein at amino like mycophenolate and mizoribine have shown antiviral acids 146–155. A peptide derived from the RSV F binding activity against the hepatitis C virus, and hence, there is domain of RhoA (RhoA77-95) was shown to inhibit RSV and scope for investigating them as potential anti-RSV drugs PIV-3 infection and syncytium formation, block cell-to-cell [143, 145]. eTh re are many other compounds that can inhibit fusion, and reduce viral titers and illness in mice. RSV replication and a well-known compound RSV 604 The fusion inhibitors are not only limited to peptide ((S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H- inhibitors; but a range of chemical inhibitors have also been benzo[e][1,4]diazepin-3-yl)-urea) showed promising results tested against RSV and benzimidazoles are well-known against RSV [146]. fusion inhibitors [175]. A lead compound was identified A derivative of antibiotic geldanamycin 17-ally-lamino- to subsequently synthesize an analogue JNJ2408068, a low 17-demethoxygeldanamycin (17AAG) and 17-dimethylam- molecular weight benzimidazole, which showed high anti- inoethylamino-17-demethoxygeldanamycin (17DMAG) tar- viral activity. It had an EC50 of 0.16 nM, 100,000 times better geted against cancer has now attracted researchers due to its than that of ribavirin. This compound showed anti-fusion antiviral property. These compounds are HSP90 inhibitors activity against RSV in a dual mode of action including and thus helpful against RSV, as RSV is dependent on heat prevention of cell-virus fusion activity as well as cell-cell Advances in Virology 15 ff ff fl Table 2: Comparison of different treatment approaches for RSV. Treatment Mechanism Example Remark References Replication inhibition RSV 604 Eective against RSV, but adverse effect on [104] Mutation Ribavirin, viramidine, merimepodib Antiviral drugs Inhibitor of inosine monophosphate the host [142–146] Ribavirin, mycophenolate, mizoribine dehydrogenase Immunostimulatory eects Ribavirin Peptide fusion inhibitors promising anti Peptide—HR121, HR212, RhoA Fusion inhibitors Inhibiting fusion protein attachment to cell RSV drug; chemical fusion inhibitors have [147–150] Chemical—BMS-433771, RFI-641 side effects Nanoparticles Inhibiting attachment to cell Silver nanoparticles, gold nanoparticles Emerging field, conclusive studies required [ 151, 152] siRNA-ALN-RSV01 Effective and safe; ALN-RSV01 completed Antisense therapy RNA interference [153–157] Phosphorodiamidate morpholino oligomers phase IIb clinical trails Plant extracts—Cinnamomum cassia, Cimicifuga foetida, Probably fusion inhibitors, Ethnobotanicals Sheng-Ma-Ge-Gen-Tang, Ginger, etc. Promising but conclusive studies required [158–164] anti-inammatory Decoctions-modified Dingchuan, Liu-He-Tang, water extract of Licorice 16 Advances in Virology fusionactivity.However,itwasfoundineeff ctiveagainstother eeff ctive against 6 laboratory and 18 clinical viruses at con- viruses of the family including HPIV-3 and measles virus. centrations between 0.008 and 0.11 mM (0.013–0.18 mg/mL). A vast screening of as many as 16,671 compounds (source The compound reduced the viral load to an extent of 1.7 logs ChemBioNet library) was conducted for anti-RSV activity in theAfrican greenmonkeymodel andalsoinmiceand in vitro and two novel compounds, N-(2-hydroxyethyl)-4- cotton rats. In order to further enhance the antiviral activity methoxy-N-methyl-3-(6-methyl[1,2,4]triazolo[3,4-a]phthal- of RFI-641, it was modified by replacing its triazine linkers azin-3-yl)benzenesulfonamide (named as P13) and the with pyrimidine [182]. However, this modification did not 1,4-bis(3-methyl-4-pyridinyl)-1,4-diazepane (named as C15) have much difference in the anti-viral activity, thus rendering were mined, which reduced the virus infectivity with IC50 this modification not much of practical use. er Th e are several values of 0.11 and 0.13𝜇 M, respectively [176]. novel nonbenzimidazole based compounds, showing anti- Recently, several groups have reported synthetic fusion RSV activity in vitro, but a more polar compound thiazole- inhibitors of RSV, especially the benzotriazole derivatives imidazole 13 was selected on the compound potency, mod- [177]. Aeft r evaluating the structure-activity relationship erate permeability, and low metabolic rate in rats, and more (SAR) of these compounds, named as series 1 compounds, detailed in vivo studies are further anticipated [183]. it wasobservedthatthe topology of thesidechainsofthese compounds is important and facilitates the modicfi ation 4.3. Nanoparticles. It has been established that metals like of their physical properties, as many of these compounds silver [184] and gold [185]haveanti-microbialactivity, but showed poor therapeutic indices (cytotoxic effects) to the cytotoxic effects of these reactive metals make them unsuit- host cells tested. In order to address these issues, a second able for their use in humans. The reactivity and behaviour of series of derivatives of the compound 1 were developed and metals canbemodulated by reducing theirsizetonanoscale. evaluated for SAR and their functionality as fusion inhibitors Carbon nanotubes (CNTs) are emerging nanomaterials for [178]. These compounds were developed from 1,which had biomedical application [186]. Polyvinylpyrrolidone (PVP) a tolerant diethylaminoethyl side chain with both polar and conjugated silver nanoparticles showed low toxicity to HEp- nonpolar functionality against RSV and had a replacement 2 cells at low concentrations and exhibited 44% RSV inhi- of the benzotriazole with benzimidazole-2-one. These were bition [151](Figure 6). Singh et al. used fusion inhibitor potent inhibitors of RSV in vitro.Thesecompounds were peptide functionalized gold nanoparticles and carboxylated named 2 and had an additional structural vector absent gold nanoparticles of size 13 nm against RSV, which showed in 1, which accounted for enhanced potency as fusion 83% and 88% inhibition of RSV, respectively [152]. Similar inhibitors and served as the base for further development approach wasemployedbyrecombinant RSVFprotein of fusion inhibitors. Further, these group 2 compounds were functionalized on gold nanorods [187]. The emergence of modified by introducing acidic and basic functional groups nanotechnology has opened new avenues for RSV treatment. into the side chains [179]. The oxadiazolone had anti-RSV activity comparable to that of ribavirin, whereas the ester modified group 2 compounds were suitable for oral admin- 4.4. Antisense Treatment. RNA interference (RNAi) which istration. These studies further led to the identification of is a normal cellular event has become a powerful means of a benzimidazole-2-one derivative called BMS-433771 which controlling gene regulation. The interference mediated by was an orally active RSV inhibitor [149]. Another compound siRNA was used against human immunodeficiency virus, studied was the 5-aminomethyl analogue 10aa with potent poliovirus, hepatitis C, and parainu fl enza virus (PIV) in cell anti-RSV activity towards BMS-433771 resistant RSV. eTh culture [188–190]. The concept of inhibiting RSV infection compound BMS-433771 was further modified at side chains using targeted antisense mechanism was applied by Jairath and with the introduction of an aminomethyl substituent et al. by silencing the RSV-NS2 gene [191]. Following the at the 5-position of the core benzimidazole moiety [180]. RNAi approach, Bitko et al. designed siRNA against the P The aminomethyl substitution in the benzimidazole ring was gene of RSV and PIV which protected mice against individual found to enhance the antiviral activity. and mixed infections upon intranasal administration [192]. Furthermore, the consecutive modicfi ation of benzimi- The effectiveness of siRNA action was observed with and dazole resulted in benzimidazole-isatin oximes which were without the use of transfection reagents. This approach was evaluated for anti-RSV activity [181]. The compound was ana- also effective when targeting the RSV-F gene [ 193]. Similar lyzed for its antiviral activity, cell permeability, and metabolic work on HEp-2 cell lines was replicated using four siRNA, stability in human live microsomes. Several other derivatives designed to silence RSV F gene, which showed inhibitory with modification such as O-alkylation and addition of action against RSV at various concentrations [194]. Silencing nitrogen atoms to isatin phenyl ring were implemented also different RSV genes too had an inhibitory action on the to enhance antiviral activity. Three compounds 18j, 18i,and RSV, a plasmid encoding siRNA which was complexed with 18n were shown to have anti-viral activity against RSV in the chitosan targeting RSV-NS1 gene decreased RSV infection BALB/c mice. Further, a compound RFI-641 was identified in BALB/c mice and Fischer 344 rats and also reduced which was found to be the most potent anti-RSV agent theassociatedinflammation [ 195, 196]. Zhang et al. showed inhibiting RSV both in vitro and in vivo andisinphase I that siRNA nanoparticle targeting RSV NS1 gene resulted in clinical trials. RFI-641 is a biphenyl triazine synthesized by increased IFN-𝛽 and IFN-inducible genes in A549 cells and coupling diaminobiphenyl to two chlorotriazine molecules in human dendritic cells, elevated type-1 IFN, and increased under microwave conditions [150]. RFI-641 was found to be differentiation of CD4 Tcells to Th1 cells[ 196]. Also Advances in Virology 17 mice treated with siNS1 nanoparticles exhibited significant Particularly in neonates, RSV bronchiolitis increases IL- decrease in lung viral titers and inflammation. 4𝛼 levels which results in increased Th2 response, so an An interferon-inducible enzyme, 2-5A-dependent RNase, antisense oligomer was synthesised for the local silencing present in higher vertebrates requires 5 -phosphorylated, of the IL-4𝛼 gene. Intranasal application of the antisense 󸀠 󸀠 2 ,5 -linked oligoadenylate (2-5A) for its endoribonuclease oligomers into a neonatal murine model reduced the Th2 activity against single-stranded RNAs. This feature of 2- type mediated pulmonary pathological signs of inflammation 5A-dependent RNase is looked upon as an effective RSV and lung dysfunction [203]. A combinatorial approach of the treatment [197]. RSV upon infection elicits immune response anti-sense oligomer against RSV and IL-4𝛼 would control of the host and particularly the interferon levels [68], and RSV infection and the adverse effects of RSV mediated this phenomenon is exploited for the anti-RSV activity of inflammation. 2-5A-dependent RNaseL. The endoribonuclease activity of Phosphorodiamidate morpholino oligomers (PMOs) are 2-5A-dependent RNaseL was used for targeting RSV M2 the oligomers where the nucleobases are covalently attached 󸀠 󸀠 gene specicfi allybycovalent2 –5 oligoadenylate target with the morpholine ring replacing the deoxyribose sugar antisense, which resulted in the reduction of RSV replication. while the phosphodiester bond is replaced by the phosphoro- The endoribonuclease activity had negligible eeff ct with an diamidate linkage [157]. Morpholino chemical modification inactive dimeric form of 2-5A linked to antisense, 2-5A linked of RNA can be used as antisense with the advantages of to a randomized sequence of nucleotides, and antisense specificity, in vivo stability, and targeted delivery. PMOs block molecules lacking 2-5A and did not affect the other RSVs the target complementary RNA and the target RNA fails or cellular RNAs [198]. Additionally, to widen the range of to interact with the proteins and thus the RNA function the approach, the effects of modicfi ation of oligonucleotides is hindered. This is specifically true for mRNA and its and RNA target sites were studied [199]. This model was translation. This phenomenon is different from antisense as improved with respect to the specificity and activity by the RNase H is not involved [204]. Hence PMOs can be a chimera of 2-5A-antisense, christened as NIH351. The designed containing the initiation codon against viruses to sequence information of RSV genome was used to develop be anti-viral. Better intake into the cell and in vivo systems NIH351 and was 50- to 90-fold more potent against RSV can be facilitated by conjugating cell penetrating peptide(s) strain A2 than ribavirin [200]. Administration of siRNA in (arginine-rich peptide (RXR) 4XB) to it. This approach was combination with ribavirin was recommended for eeff ctive attempted against the RSV-L gene to inhibit RSV in cellular treatment [201]. The parent molecule was chemically modi- and murine models [205]. fied to further increase the in vivo stability and specicfi ity and potency of NIH351. The resulting new version RBI034 was 4.5. Ethnobotanicals. Various natural and synthetic chemical ∼50% more eeff ctive than the parent molecule against RSV compounds have been screened for their application to (strain A and B) and was not cytotoxic in the effective dose treat RSV infections. Plants are rich sources of alkaloids, ranges. RBI034 treatment of African green monkeys shows steroids, flavonoids, and other complex compounds that have promising results [202]. medicinal value and medicinal plants contribute significantly Alvarez and coworkers [153]cameupwithanewRSV- to the traditional Indian and Chinese medicine. Ancient NS1 gene specific siRNA (ALN-RSV01) having a broad Chinese literature has descriptions of plant extract against spectrum of antiviral activity that targeted the nucleocapsid respiratory diseases [206]. The exact active compound or gene of RSV. In vivo BALB/c murine studies demonstrated action of the traditional formulations is not understood that intranasal dosing of ALN-RSV01 resulted in a 2.5- to [207]. Now, modern assays have made it possible to get an 3.0-log-unit reduction in RSV lung concentration. To scale insight into the mechanism and purification of active plant up this molecule for RSV treatment in humans, the safety, based compound. Extracts of Lonicera deflexicalyx (Chin tolerability, and pharmacokinetics were tested on healthy Jinyinhua) were tested against RSV. The active compound adults, demonstrating its safety and tolerance in human from the extract was 3, 5-dicaeff oylquinic acid (CJ 4-16- subjects [154]. In the human clinical trials of ALN-RSV01, 4), which was isolated, and purified by a series of chro- healthysubjectsweregrouped andadministeredeithera matographic processes. It is suggested that CJ 4-16-4 is a placeboorALN-RSV01 nasalspray forRSV.Therewas 44% fusion inhibitor and the in vitro and in vivo studies suggest reduction in the RSV infection in the subjects who received that it is a more eeff ctive RSV inhibitor than ribavirin ALN-RSV01 without any adverse effect. u Th s, this study in [208]. Cytopathic effect (CPE) assay based screening showed real terms has established a unique “proof-of-concept” for an that 27 of 44 herbs had moderate or potent anti-RSV RNAi therapeutic agent in RSV treatment [155]. ALN-RSV01 activity [206]. The plant extracts from Cinnamomum cas- proved to be safe and was eecti ff ve against RSV even in a sia, Cimicifuga foetida, Sheng-Ma-Ge-Gen-Tang (SMGGT) complex clinical situation like lung transplants, which was a (Shoma-kakkon-to), Xiao-Qing-Long-Tang (Sho-seiryu-to, remarkable achievement [156]. It is the product of Alnylam so-cheong-ryong-tang), Ge-Gen-Tang, and Ginger (Zingiber Pharmaceuticals, Inc. and has completed phase IIb clinical ocina ffi le) show anti-RSV activity. These extracts probably trials. inhibit RSV infection by blocking the F protein binding to the Another approach of RNAi treatment to combat RSV cell and some of the extracts even stimulate IFN-𝛽 production is to decelerate the adverse effects of RSV mediated [158–163]. Some decoctions like modified Dingchuan (con- Th2 type immune response because the aggravated host sists of Salviae miltiorrhizae radix, Scutellariae radix, Farfarae immune response is more harmful than RSV infection itself. flos and Ephedrae herba), Liu-He-Tang (consist of 13 plant 18 Advances in Virology extract), and water extracts of Licorice (Radix glycyrrhizae of the respiratory viruses [6, 213]. The situation is more and Radix glycyrrhizae Preparata) have shown eeff ctiveness intricate as some authors report that coinfection with non- against RSV in vitro. Moreover, the modified Dingchuan RSV respiratory viruses tends to increase RSV severity [214] decoction (MDD) exhibited anti-inflammatory and anti-viral and also there is a hypothesis of a synergistic association of eect ff in mice (SPF ICR mice) infected with RSV. MDD RSV with other viral or bacterial infections [215]. u Th s, this suppressed eotaxin, IL-4 and IFN-𝛾 level in serum, and topic is a subject of debate and these contradictions need mRNA expression of TLR4 and NF-𝜅 B in lungs of RSV claricfi ation and consensus for proper treatment options. A infected mice [164]. link between atopy, asthma, and RSV was suspected for a very long time, but now there is supporting evidence for this possible relationship [216–218]and is also ascribed partly to 5. Challenges in the Diagnosis, genetic factors of RSV and the host [70, 71, 219, 220]. The Prevention, and Treatment of RSV activity of RSV in the community may be aeff cted by many Technology has provided enough capabilities for the detec- factors, including climate, air pollution [221], race/ethnicity tion of RSV in various sample types at various stages of [222], and social behavior of the population. Under these infection using an array of techniques, but the challenge is, complex factors associated with RSV, it is established that availability of these facilities at the correct time for a reason- early detection of risk factors and medical intervention can reduce the incidence of RSV [223]. A balance of execution able cost. eTh most important perspective of RSV diagnosis is the strategic management of choice between the point and/orabeyanceofprophylacticmeasuresiscriticalwith of care testing and central laboratory testing [36]. Though respect to the above discussed determinants [224]. There is limited data to correlate RSV global transmission dynamics the point of care testing gives rapid detection advantage, it sueff rs from lower sensitivity and thus is a problem to with climate and population [2], due to which it is difficult to be dealt with. Rapid diagnostic tools like RT-PCR/ESI-MS, develop strategies for RSV prevention and treatment. microarray based semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus 6. Conclusions nucleic acid test SP (RVNATSP) (Nanosphere, Northbrook, IL) have proved their efficiency, but their application in Currently, there is no vaccine or effective treatment against routine clinical practice is still a challenge. RSV, but the rapid and sensitive RSV detection is possible. There are numerous molecules that can be potential eTh detection techniques are ameliorated by incorporating antiviraldrugs,but thescreening of avastnumberof one or more methods and with the advancement in material compounds is cumbersome; hence, high throughput filtering science and biophysical capabilities, it has reinforced the is an essential part of drug development. As an example, development and design of RSV detection systems. However, when 313,816 compounds from the Molecular Libraries Small an eecti ff ve detection technique can be transformed into Molecule Repository were screened against RSV in HEp-2 effective diagnosis by integrating it into the community cell line, only 409 compounds showed 50% inhibition of the health monitoring program at a reasonable cost. Prevention cytopathic effects [ 209]. The challenge after this sophisticated of RSV infection at present is limited to only high risk screening is the translation into drugs by clearing the phases individuals with a limited ecffi acy. New preventive measures of in vivo animal studies and human trials. A setback for research like DNA vaccines, subunit vaccines, and nano- the development of therapies against RSV is the lack of a vaccineshavereached animal trials.Onthe otherhand, good animal model as they do not truly manifest effects of the RSV treatment approaches using antisense oligomers, RSV infection as in humans. RSV experiments in various fusion inhibitors, and benzimidazole drug have proceeded animal models like BALB/c mice, cotton rats, macaques, into clinical trials. eTh challenges associated with RSV African green monkeys, owl monkeys, cebus monkeys, bon- management are categorically numerous. However, at the net monkeys, olive baboons, and chimpanzees are evident current pace of scienticfi research and development and with in the literature. Small animal models like BALB/c mice the implementation of scientific, commercial, and program andcottonratsare commonly used duetoeaseofhandling recommendations to develop epidemiological strategies, it and low cost, whereas the primate studies are conducted seemsoptimistictohaveaneeff ctivediagnosis,prevention, with more stringent regulations and bear heavy expenses and treatment solution for RSV in near future. [210, 211]. eTh re are many aspects that need to be addressed in the challenges for vaccine development programs and Competing Interests the technological interventions to deal with RSV [212]. These challenges include safety issues concerning the subjects eTh authors declare that they have no competing interests. involved in clinical trials, as evident by the failure of formalin inactivated RSV vaccine and motavizumab at the clinical trial levels which resulted in undesired immunogenic responses in Acknowledgments the patients involved [34, 116]. Though the present data conclude that RSV is one of the The authors acknowledge the National Science Foundation leading causes of morbidity and mortality in children and Grant NSF-CREST (HRD-1241701) and HBCU-UP (HRD- elders, there is no significant correlation between increased 1135863). They thank Eva Dennis for the gur fi es used in this disease severity, respiratory deaths, and detection of any paper. 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Recent Advances in Diagnosis, Prevention, and Treatment of Human Respiratory Syncytial Virus

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Hindawi Publishing Corporation Advances in Virology Volume 2013, Article ID 595768, 26 pages http://dx.doi.org/10.1155/2013/595768 Review Article Recent Advances in Diagnosis, Prevention, and Treatment of Human Respiratory Syncytial Virus Swapnil Subhash Bawage, Pooja Munnilal Tiwari, Shreekumar Pillai, Vida Dennis, and Shree Ram Singh Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL 36104, USA Correspondence should be addressed to Shree Ram Singh; ssingh@alasu.edu Received 31 July 2013; Accepted 30 September 2013 Academic Editor: Subhash Verma Copyright © 2013 Swapnil Subhash Bawage et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Human respiratory syncytial virus (RSV) is a common cause of respiratory infection in infants and the elderly, leading to significant morbidity and mortality. eTh interdisciplinary fields, es pecially biotechnology and nanotechnology, have facilitated the development of modern detection systems for RSV. Many anti-RSV compounds like fusion inhibitors and RNAi molecules have been successful in laboratory and clinical trials. But, currently, there are no eeff ctive drugs for RSV infection even aer ft decades of research. Eeff ctive diagnosis can result in eeff ctive treatment, but the progress in both of these facets must be concurrent. The development in prevention and treatment measures for RSV is at appreciable pace, but the implementation into clinical practice still seems a challenge. This review attempts to present the promising diverse research approaches and advancements in the area of diagnosis, prevention, and treatment that contribute to RSV management. 1. Introduction Myanmar, aTh iland, Madagascar, Kenya, Zambia, Nigeria, and Columbia. The data about human RSV described in Worldwide, there are reportedly about 12 million severe literature over the years seem to have been unchanged and 3 million very severe cases of lower respiratory tract significantly, indicating the severity of RSV and the urgent infection (LRTI) in children [1]. Respiratory syncytial virus concern to address this issue. An estimate of more than 2.4 (RSV) is a common contributor of respiratory infections billion US dollars per year is the economic cost of viral lower causing bronchiolitis, pneumonia, and chronic obstructive respiratory tract infection in children [6]. pulmonary infections in people of all ages but affects mainly RSV is a Paramyxovirus belonging to the genus Pneu- children and elderly along with other viral infections leading movirus. RSV is an enveloped, nonsegmented, negative, to high mortality and morbidity [2–4]. A recent global survey single stranded linear RNA genome virus (Figure 1). RSV suggests that RSV is not prevalent throughout the year in genome (∼15 kb) has 10 genes encoding 11 proteins with the tropical regions of the globe, but the incidence peaks in twoopenreading frames of gene M2 [7, 8]. Other genes winter with a wide ranging persistence depending on the geo- include nonstructural proteins NS1 and NS2 (type I inter- graphical topology [5]. RSV has been reported to be a preva- feron inhibitors), L (RNA polymerase), N (nucleoprotein), P lent lower respiratory tract pathogen distributed worldwide (Phosphoprotein cofactor for L), M (Matrix protein), M2.1 including countries from both, the developed and developing and M2.2 (required for transcription) SH (small hydrophobic world. The major countries with RSV seasonal outbreaks protein) G (glycoprotein), and F (fusion protein). Being a include USA, Canada, Cambodia, Mexico, Uruguay, Brazil, negative strand RNA genome virus, RSV packages its own Peru,France, Finland, Norway,Sweden,Latvia, Denmark, polymerase into the nucleocapsid. Of these proteins, fusion Germany, Netherlands, Ireland, Italy, Turkey, Iran, Saudi protein (F) is indispensable for viral attachment to the host Arabia, Australia, New Zealand, China, Korea, Hong Kong, and entry into the host cell. Although the G protein is Japan, India, Pakistan, Bangladesh, Nepal, Taiwan, Vietnam, responsible for the preliminary attachment, the F protein is 2 Advances in Virology NS1 NS2 SH M2 L protein Single stranded RNA N protein P protein Matrix protein SH protein F protein G protein (a) (b) Figure 1: Structure and genome organization of respiratory syncytial virus. (a) Approximately 200 nm RSV virion particle and (b) single stranded negative RNA genome consisting of 10 genes. necessary for the fusion, budding, and spread of the virus as carriersofthe virusfor 1to3weeks. RSVinfection [9, 10]. After attachment to the host cell, RSV fuses with in children of nosocomial origin is associated with higher the host cell membrane using the F protein through the mortality than community-acquired illness because of the 6 helix coiled-coil bundle of the F protein, a mechanism pre-existing morbidity [14, 15]. Severe RSV disease risk hovers characteristically found in paramyxoviridae members [11]. forthe elderly andadultswithchronic heartorlungdisease Although the detailed mechanism of RSV infection is not or with weakened immune system [16]. RSV infection does fully understood, the most accepted mechanism is the entry not provoke lasting immunity [17] therefore, reinfection is of the nucleocapsid into the host cell mediated by the F very common [18]. Recently, RSV infection was reported to protein through clathrin mediated endocytosis [12]. The account for hospitalizations and mortality in elderly people RNA is rst fi converted into a plus strand, which serves as [19]. RSV accounted for severe lower respiratory tract infec- the template for replication; whereas for transcription, the tions including chronic lung disease, systemic comorbidities, RNA genome itself transcribes mRNA for protein synthesis and even death. At present, there is no specicfi treatment without any intermediate. for RSV infection ever since its rfi st discovery in 1956 [ 20]. Almost all children of 2 years of age will have had an RSV Currently, Food and Drug Administration (FDA) approved infection and leading to 160,000–600,000 deaths per year [4]. prophylactic drug for RSV that includes palivizumab and Approximately, 25% to 40% of infants and children at the ribavirin; administered along with symptomatic treatment first exposure to RSV have signs or symptoms of bronchiolitis drugs and supportive care. Currently, techniques used for or pneumonia. These symptoms include rhinorrhea, low- diagnosis of RSV include ELISA, direct immunouo fl rescence, grade fever, cough, and wheezing. The symptoms in adults western blot, PCR, and real-time PCR. eTh diagnosis and may include common cold, with rhinorrhea, sore throat, treatment scenario has signica fi ntly changed with the advent cough, malaise, headache, and fever. It can also lead to of advanced techniques and in-depth understanding of RSV exacerbated symptoms such as severe pneumonia in the biology, but the execution of these clinical developments elderly, especially residing in nursing homes [13]. Usually, in practice requires extensive study and time. This review children show symptoms within 4 to 6 days of infection presents the recent advances in the diagnosis, prevention, and andmostofthemrecover in 1to2weekswhile serving treatment of RSV (Figure 2). Advances in Virology 3 DNA based assays Immunoassays Nanotechnology based assays Immuno-PCR Real-time PCR ELISA Quantum dots Diagnosis LAMP LFIA Microarray PCR-ESI-MS OIA Live cells RNA imaging DFA Microarray SERS DNA vaccines Nutrition Antibodies Prevention Subunit vaccines Nanovaccines Anti-viral drugs Fusion inhibitors Antisense RNA Treatment Nanoparticles Ethnobotanicals Figure 2: A schematic representation of RSV management through coordinated diagnosis, prevention, and treatment. 2. Diagnosis also caused urticaria [34]. It is presumed to be a better bindingtargetthanthe conventional Fprotein ELISA. This There are ten known genotypes of RSV based on the sequence method could prove more eecti ff ve than the F protein analysis of the RSV genome, which suggests that the pathogen ELISA due to its higher sensitivity to the degradation of changes with time and the resulting genotypes pose a threat motavizumab. In a simple thin layer amperometric enzyme to the public health. Information from comparative genome immunoassay, RSV was detected as early as 25 minutes, at analysis has been utilized for evolutionary studies and most low cost with comparative sensitivity to that of real-time importantly for the development of detection and treatment PCR and immunou fl orescence assays [ 35]. The assay is based studies [21, 22]. Diagnostic assays for detection of pathogens on the development of a double layer sandwich method have a pivotal role in public-health monitoring [23]. However, similar to ELISA. It involves a polystyrene microarray slide diagnosis becomes difficult when the causative agent exhibits coated with monoclonal antibody which captures the antigen overlapping symptoms with other disease(s) or remains (RSV). The antigen-antibody complex is detected by horse nonsymptomatic. Hence the correct diagnosis has to be radish peroxidase conjugated secondary antibody on a screen empirically derived for the treatment. Some of the promising printed electrochemical cell coated with the substrate. molecular and biophysical techniques for RSV diagnosis are discussed below (Table 1). 2.1.2. Immunofluorescence Assay. Presently, immunou fl ores- cence is one of the most common and rapid RSV detection 2.1. Immunoassays techniques used, where the antigen is detected by a u fl o- 2.1.1. Enzyme-Linked Immunosorbent Assay. Enzyme-linked rescently tagged antibody. Direct fluorescent antibody assay immunosorbent assay (ELISA) is the enzyme facilitated col- (DFA) is a standard detection technique used for decades orimetric detection of specific protein-antibody complexes. and other RSV detection techniques are oeft n compared ELISA is extensively used for detection of various proteins at to DFA for evaluating their ecffi iency [ 24]. An indirect very low concentration in different sample types, thus making assay which uses secondary u fl orescently tagged antibody is it clinically significant in routine diagnosis of pathogens. another option to DFA. Currently, there are many molecular ELISA for RSV detection is mainly based on targeting RSV techniques that are more sensitive and reliable than DFA, F protein (antigen). Recently, several modifications of the but DFA is widely used for RSV detection in clinical samples classical ELISA technique have been ecffi iently developed due to the ease and rapidity. eTh sensitivity and specicfi ity of and employed for the detection of RSV. Sensitivity of ELISA DFA are a subject of variance as the success of the technique was increased by using the high affinity anti-RSV F antibody is dependent on numerous factors, mainly the skills of the peptides derived from the motavizumab [85]. Motavizumab technician and nature of the sample. A study showed that is a high affinity antibody based therapeutic against RSV DFA could detect RSV with a sensitivity and specificity of which binds to RSV F protein; however, it was disapproved by 77.8% and 99.6%, respectively (positive predictive value was theFDA duetohigherhypersensitivityinpatientsreceiving 98.6%and thenegativepredictivevalue was94%). DFAis motavizumab as compared to palivizumab. Motavizumab a reliable technique for RSV detection for patients tested 4 Advances in Virology ff ff fi fl fl Table 1: Comparison of RSV detection techniques. Technique Reference Principle Advantages Drawbacks Current usage status (A) Fluorescence based methods Microscopic detection of RSV with Research intent, Hospital based (1) DFA [24, 25] specific antibody conjugated with Easy procedure Human error, fading of dyes procedure, commercial fluorophore. diagnostic assays Detection of signals from fluorescent nanoparticles upon encounter with RSV Photostable, inorganic in nature, resistant Research intent (2) QDs [26–31] Toxicity, insolubility either through microscopy or ow to metabolic degradation cytometry (3) Molecular Hairpin DNA functionalized gold Probable gene silencing, beacon based [32, 33] nanoparticle with fluorophore Live cell imaging with real-time detection Research intent metabolic degradation imaging hybridization with target mRNA (B) Immunoassays Specific binding and colorimetric Easy protocol, high specificity and Cumbersome, prone to human Hospital based procedure, (1) ELISA [34, 35] detection of antigen-antibody complex sensitivity errors commercial diagnostic assays Presence of specific antigen-antibody complex formed alters the reective Needs confirmation by other Research intent, not (2) OIA [36–38] Easy, rapid, specificity, cost effective surfaces properties which is visually tests for negative samples commercialized detected Immuno-complexes detected Easy,rapid,handy,costeective, FDA Nonquantitative, limit of sample Hospital based procedure, (3) LFIA [39–42] chromatographically approved volume limits detection commercial diagnostic assays (C) Molecular methods Colorimetric/turbidimetric detection of Semiquantitative, designing Research intent, not (1) LAMP [41, 43–45] isothermal amplification of DNA using Sensitivity and specicity compatible primer set commercialized specific primer Amplification of viral cDNA and Rapid and sensitive than conventional Research intent, hospital based (2) PCR [46, 47] High limits of detection visualization of PCR product culture methods procedure (3) Real-Time Real-time amplification of target DNA or Rapid (3–5 hours), highly sensitive and Research intent, hospital based [48–53] Expensive PCR cDNA very low limits of detection procedure, commercial assay (4) Multiplex Simultaneous detection of multiple Research intent, hospital based [54–57] Use of multiple primer and/or probe sets Less sensitive PCR pathogenic species or strains procedure Very low limits of detection, improved A combination of immunoassay and Research intent, not (5) Immuno-PCR [58, 59] limits of detection over individual ELISA, Complex experimental design real-time PCR commercialized and PCR (4000 and 4 fold. respectively) Hybridization of sample biomolecules to Highly sensitive, large scale identification Research intent, hospital based (6) Microarray [60–74] immobilized target DNA or protein on a of multiple pathogens; protein and Cost-ineective procedure, commercial assay chip nucleic acid targets (D) Biophysical method Highly sensitive and specific even at Mass spectroscopy of PCR-amplicons Research intent, not (1) PCR-ESI-MS [75, 76] strain level and efficient multiple Expensive through electron spray dispersion commercialized pathogens detection. Inelastic scattering of monochromatic radiation upon interaction with an Rapid and nondestructive detection of Research intent, not (2) SERS [77–84] Sample preparation analyte with low-frequency vibrational analytes with high sensitivity commercialized and/or rotational energy Advances in Virology 5 within first 3 days aeft r onset of symptoms, but the sensitivity pathogens like RNA viruses, DNA virus, or bacteria [43]. decreases if tested 4–7 days aer ft the onset of the symptoms Although this method is rapid, the requirement of the [24]. DFA can detect RSV alone or as a multivalent test turbidimeter makes it slightly inconvenient as a cost-effective for other respiratory viruses (influenza A and B viruses, detection technique. Development in the chemistry of the parainfluenza virus types 1 to 3, and adenovirus) using LAMP has made it possible to get rid of the turbidimeter as SimulFluor Respiratory Screen assay [25]. it is now possible to visually detect the presence of pathogen in the test sample. LAMP is now referred to as colorimetric detection of loop-mediated isothermal amplification reaction 2.1.3. Optical Immunoassay. The direct visualization of an due to incorporation of the dye in the chemistry to aid visual antigen-antibody complex often referred to as optical immu- detection. Wang et al. (2012) have developed such an RT- noassay (OIA) is used for qualitative detection of Streptococ- LAMP assay for the detection of human metapneumoviruses cus and influenza virus. Visualization of immunocomplexes (which include rhinovirus, RSV, influenza virus A/PR/8/34 canbeenhancedbyimmobilizingthemonaspecialreflecting (H1N1)), which can detect as low as ten viral RNA copies surface. eTh antigen-antibody complex forms a thin layer, with better efficiency than RT-PCR [ 44]. It is now possible which changes the reflective properties of the surface [ 36]. to detect RSV A and B strains in nasopharyngeal specimen The technique is simple, rapid, and sensitive and as low as using multiplex LAMP (M-LAMP) in just 30 minutes. M- 1 ng of antibody per mL can also be detected [37]. OIA is LAMP had a sensitivity and specificity of 100% when com- now used for detection of RSV with a sensitivity, specicfi ity, pared with PCR [45]. positive, and negative predictive values of 87.9%, 99.6%, 98.9%, and 94.5%, respectively. Though OIA oer ff s rapid and cost-eeff ctive RSV detection, it is recommended that negative 2.3. Polymerase ChainReaction(PCR) BasedDetection results of OIA should be conrfi med by other tests [ 38]. 2.3.1. Conventional PCR. The applicability of PCR is ubiqui- tous andhas made thediagnosis of pathogenstremendously 2.1.4. LateralFlowImmunoassay. Lateral flow immunoassay rapid and sensitive [46]. RSV is usually challenging to detect (LFIA) is an immunochromatographic technique known for due to poor viral titer and sensitivity to antigen based detec- the rapid detection of RSV from nasal washes or nasal tion methods. u Th s, a PCR based method was developed and aspirates. The lateral flow of antigen-antibody complex on compared to serological and culture based detection methods the substrate matrix reaches the reaction area and results in in adults having respiratory infections [47]. The PCR method formation of colored band indicating the presence of antigen was based on the reverse transcription-nested PCR technique in thespecimen. eTh re aremanyLFIAkitslikeRemel Xpect, involving the outer and inner primers designed from the Binax Now RSV, BD Directigen EZ RSV, QuickLab RSV Test, F gene of RSV strain A, over a two day procedure with a and RSV Respi-Strip [39–42]. eTh sensitivity and specicfi ity sensitivity of 73%. This method is faster as compared to the are normally above 90% and 95%, respectively, but vary as conventional culture method which usually takes 3–5 days, per the manufacturer. Modifications of LFIA may include resulting in faster treatments. colloidal gold conjugated with antibody specific to RSV in thematrixfor trapping theantigen andassisting thegold mediated reaction for color band development. 2.3.2. Real-Time PCR. Although conventional reverse tran- scriptase-PCR (RT-PCR) is sensitive as compared to the 2.2. Loop-Mediated Isothermal Amplification. Loop-mediat- culture methods, it suffers from the lower sensitivity. This ed isothermal amplification (LAMP) is a nucleic acid based problem has been obviated by the development of real-time detection method used for bacterial and viral pathogens. It PCR based methods. Real-time PCR is by far the most can be used for RNA viruses where the additional step of sensitive method for the detection and diagnosis of a wide cDNA synthesis is required and commonly designated as array of pathogens, including RSV. Several studies have been reverse transcription LAMP (RT-LAMP). LAMP consists of conducted for the development of real-time PCR assays for pairs of primers specific to cDNA, which is then amplified by RSV detection, especially the RT-PCR. In one such study, autocycling strand displacement activity of DNA polymerase a rapid, sensitive, and specific method was devised based generally at 60 C. eTh reaction time of 1 to 1.5 hours is on TaqMan real-time PCR for the detection of both RSV A sucffi ient and can be monitored by real-time turbidimeter and RSV B strain [48]. The primer and the probe sets were and the resulting product can also be viewed by agarose designed from the nucleocapsid gene (N). The sensitivity of gel electrophoresis. This method can distinguish RSV strain this method was found to be 0.023 PFU (plaque forming unit) Aand Buponrestriction digestionofthe product[41]. or two copies of mRNA for RSV A, whereas the sensitivity Alternatively, RSV strain A and B can be detected designing for RSV B was 0.018 PFU or nine mRNA copies. This method specific primer sets for them. eTh efficiency of RT-LAMP is fast and efficient as the diagnosis was performed within 6 was tested for RSV detection from nasopharyngeal aspirates hours of the procurement of samples. and compared with viral isolation, enzyme immunoassay, A similar assay based on real-time PCR was developed immuno-chromatographic assay, and real-time PCR. RT- and employed in the detection of RSV in the bronchoalveolar LAMP was the most sensitive among all the methods tested, lavage (BAL)oflungtransplantpatientsorpatientswith with the exception of real-time PCR. Also, the RT-LAMP was respiratory infections [49]. The assay was designed based on specific for RSV and did not react with any other respiratory theRSV Ngeneand rfi st involved ascreening step of theRSV 6 Advances in Virology positive samples using a SYBR green based assay, followed 2.4. Microarray. Correlating gene expression signatures with by quantitative real-time PCR using TaqMan based assay to disease progression of the patient’s individual genetic profile be more cost eeff ctive. Also, the assay was developed for the is possible by comprehensive understanding and interpre- detection of both RSV A and B subgroups separately. The tation of the dynamics of biome-interactions in the lungs. assay was found positive in 16% of transplant patients, thus This would result in more efficient therapy for respira- indicating some possible association between RSV infection tory diseases via the concept of personalized medicine, and lung transplant tolerance. wherein the microarray finds its applications. Microarrays Real-timePCR basedonRSV Ngenehas also been used are the miniaturized assay platforms with a high-density to quantify RSV from the nasal aspirates of children [50]. array of immobilized DNA or protein. Hybridization of The method was one log more sensitive than the conventional sample biomolecules to corresponding DNA or protein on culture method. The real-time PCR assay resulted in 56% of the chip is detected, which allows determination of a variety positives, whereas the immunou fl orescence assay had 41.3% of analytes present in the samples in a single experiment and culture method had 45.3% of positives. Surprisingly, the [60]. Microarray has proved to be a robust and reliable RSV to GAPDH ratio did not differ in children with severe or tool to understand the echelon of genomics, transcriptomics, nonsevere infection, raising confusion about the correlation and proteomics. Implementation of microarray with the of viral load to the severity of infection due to the intensity metagenomic approach serves for rapid virus identifica- of viral replication, genetic susceptibility of the host and tion. Microarray based identification and characterization immune responses, thus, making it necessary to consider of viruses in clinical diagnostics are possible by designing other factors besides the viral load. oligonucleotide probes using the sequence data available in A similar assay was developed for the detection of RSV the public database. This approach is capable of discovering using primer-probe sets from the RSV F gene [51]. A com- novel viruses, even though there are no conserved genes that parative analysis was made between the nested RT-PCR, real- can be targeted by sequencing. In the case of respiratory time PCR, and ELISA, wherein real-time PCR was 25% more diseases, it can be executed by collecting the bronchoalveolar sensitive than the conventional nested PCR, thus making it lavage enriched by nuclease treatment followed by filtration more applicable for clinical samples. Additionally, the real- and extraction of total nucleic acids. Further, the nucleic acids time PCR was performed in two steps to increase the sensitiv- are amplified by a random priming-based method, referred to ity of the samples. This method was more rapid as the results as sequence independent single primer amplification, giving were obtained in 3.5 to 4 hours upon receiving the samples. near-full-length reads of genomes of RNA or DNA viruses, Several other reports also emphasize the applicability of real- which could then be compared to known viruses and used time PCR in detection of RSV in immunocompromised for designing the oligomers for the microarray [61]. u Th s, patients [52]. Besides the RSV F and N gene, the RSV matrix sequencing coupled with microarray is a powerful high gene andpolymerasegenehavealsobeenusedtodetectRSV throughput diagnostic tool. in children [53]. The latter method had the ability to quantify The utility of a metagenomics based strategy for broad- and classify RSV with high efficiency and rapidity. However, spectrum diagnostic assay using microarrays was demon- the sensitivity of the technique relies on the age of the strated for screening viruses like Rhino virus, Parainfluenza patients. virus, Sendai virus, Poliovirus, Adenovirus, and RSV from clinical samples [62]. A well-known example is “Virochip” which is a pan-viral microarray, designed to simultaneously detect all known viruses, and has comparable or superior sen- 2.3.3. Multiplex PCR. The multiplex PCR approach has been used for the detection and subtyping of RSV and human sitivity and specificity to conventional diagnostics [ 63, 64]. influenza virus simultaneously [ 54] and as many as 18 Similarly, an influenza microarray, “FluChip-55 microarray”, respiratory viruses could be detected [55]. The method was for the rapid identification of influenza A virus subtypes based on the primer sets of hemagglutinin and nucleoprotein H1N1, H3N2, and H5N1 was developed [65]. The procedure is simple and follows few steps including RNA extraction from gene of influenza virus and nucleocapsid protein gene of RSV and found to be rapid, specific, and sensitive. However, clinical samples, subsequently their reverse transcription, the method suffered from the drawback that it could not second-strand cDNA synthesis, and then PCR amplification of randomly primed cDNA. eTh hybridization of nucleic distinguish between RSV A and B. This method could prove significant during a respiratory outbreak for surveillance. In acidswithprobesisdonebyfluorescentdyesorbyan such situations, multiplex real-time PCR assay, such as the alternative method of electrochemical detection. eTh latter commercial “Simplexa Flu A/B & RSV kit” that differentiates method relies on a redox reaction to generate electrical influenza A virus, influenza B virus, and RSV, is very useful current on the array for measurement. Incorporation of Cy3 [56]. Likewise, recently, a rapid and sensitive detection assay u fl orescent dye and hybridization to the microarray [ 66] for RSV and other respiratory viruses was developed by has been empowered by specific algorithms to match the Idaho Technologies christened as FilmArray (Idaho Tech- diagnostic needs, leading to the final and critical step of scanning and analysis [67]. nologies, Salt Lake City, UT). This is an automatic real-time molecular station with capability of nucleic acid extraction, The genes involved in the pathways of neuroactive ligand- initial reverse transcription, and multiplex PCR followed receptor interaction, p53 signaling, ubiquitin mediated prote- olysis, Jak-STAT signaling, cytokine-cytokine receptor inter- by singleplex second-stage PCR reactions for specific virus detection [57]. action, hematopoietic cell lineage, cell cycle, apoptosis, and Advances in Virology 7 cancer were upregulated in RSV-infected BEAS-2B cells. RSV mass spectrometry (Py/GC/MS), Capillary electrophoresis- infection up-regulated 947 and 3047 genes at 4 h and 24 h, MS, and Liquid chromatography mass spectrometry (LC- respectively, and 124 genes were common at both instances. MS) are the few examples that are used for the identification Moreover, 1682 and 3771 genes were downregulated at 4 h and of pathogens [89]. But the MS technique that promises 24 h, respectively, and only 192 genes were same. Respiratory robust practical application for pathogen detection is the disease biomarkers like ARG2, SCNN1G, EPB41L4B, CSF1, electrospray ionization mass spectrometry (ESI-MS) [92, 93] PTEN, TUBB1, and ESR2 were also detected. RSV infection Figure 3(a). signs and symptoms render a partial consequence of host A notable ESI-MS for global surveillance of influenza pathogen interaction, but the transcription profile is better virus was accomplished by Sampath et al. [75]bycoupling exemplified by microarray. eTh transcription profiles of RSV MS with reverse-transcription PCR (RT-PCR). eTh study cor- infected mice lungs and lymph nodes showed gene expression rectly identiefi d 92 mammalian and avian influenza isolates of antigen processing and inflammation. The response is (which represented 30 different H and N types, including 29 higher in lungs on the day 1 aer ft RSV infection than day 3. avian H5N1 isolates). eTh analysis showed more than 97% The gene expression profile shortly aer ft RSV infection can be sensitivity and specificity in the identification of 656 clinical accountedasabiomarkerandcanbescaledupfordiagnostics human respiratory specimens collected over a seven-year with in vitro and in vivo profiles [ 68, 69]. period (1999–2006) at species and subtypes level. The surveil- Sometimes DNA microarrays do not confer their utility lance of samples from 2005-2006 influenza virus incidence in very specicfi investigations in the case of personalized showed evidence of new genotypes of the H3N2 strains. eTh medicine wherein the manifestation of disease occurs at the study also suggested approximately 1% mixed viral quasi- transcription level. This situation may arise in complications species in the 2005-2006 samples providing insight into viral of RSV with other associated disorders like asthma [70, evolution. This study led to a number of RT-PCR/ESI-MS 71]. As protein is the abundant functional biomolecule, based detection methods for RSV and related respiratory reflecting the physiological or pathological state of the organ, viruses like influenza A and B, parainfluenza types 1–4, protein microarray profile is an option to access under adenoviridae types A–F, coronaviridae, human bocavirus, and this situation [72]. Protein microarray has analytical and human metapneumovirus screening [76]. These assays had functional applications to study the protein-protein, protein- 87.9% accuracy, compared to conventional clinical virology DNA, and protein-ligand interactions. es Th e features enable assays and pathogens undetected by traditional clinical virol- the profiling of immune responses and are thus important ogy methods could be successfully detected. The advantage for diagnostics and biomarker discovery. Protein microarray of RT-PCR/ESI-MS platform is the ability to determine the can serve as a rapid, sensitive, and simple tool for large-scale quantity of pathogens, detailed pathogen characterization, identification of viral-specific antibodies in sera [ 73, 74]. An and the detection of multiple pathogens with high sensitivity extensive study was done during the 2002 SARS pandemic and ecffi iency. using coronavirus protein microarray to screen antibodies in human sera (>600 samples) with>90% accuracy and at least 2.6. Nanotechnology Based Detection. Recent advancements as sensitive as, and more specific than the available ELISA in nanotechnology have changed the perception and perspec- tests [73]. u Th s, this system has enormous potential to be used tive of research.Whenscaleddowntonanometers, theprop- as an epidemiological tool to screen viral infections. erties of matter change and nanotechnology exploits these new properties and harnesses them with the existing tech- 2.5. Mass Spectroscopy. Mass spectroscopy (MS) has become nologies to exceptional capabilities. Techniques discussed a method of choice for molecular investigation of pathogens below are examples of the nanotechnology based detection as the reliability is reinforced due the well-characterized approaches which utilize basic traditional but indispensable sequence information of nucleic acids or proteins and even detection techniques. for intact viruses [86, 87]. But pragmatic usage of MS is possible when coupled with various chromatography and 2.6.1. Nanoparticle Amplified Immuno-PCR. Immuno-PCR, affinity-based techniques. eTh combination of affinity based a combination of ELISA and PCR, is used widely for the viral detention and nucleic acid based MS serves as a solution detection of various bacterial and viral antigens with lower for low detection limits. Affinity-based methods employing titers as meagre as zepto moles [58]. Perez et al. [59]reported nanotechnology can be used to nd fi traces of target pathogen a modification of immuno-PCR using gold nanoparticles to improve detection limits. PCR amplification of pathogen for RSV detection. Target extraction was enhanced by using nucleic acid combined with MS can be used as substitutes magnetic microparticles (MMPs) functionalized with anti- [88–90]. MS has the advantage of rapid identification of mul- RSV antibody to capture the antigen (RSV). The MMP- tiple viruses at the same time and even identifies the protein RSV complex is then countered with gold nanoparticles modification status [ 86, 91]. Now, MS is no longer confined functionalized with both, palivizumab (Synagis), an anti-RSV to proteomics based analysis, and MS based genomics have F protein antibody, and DNA sequence partially hybridized become common practice. er Th e are several variations of with a tag DNA sequence (fAuNP). eTh MMP-RSV-fAuNP MS, like Matrix-assisted laser desorption/ionization mass complexisthenheatedtorelease thepartially hybridized spectroscopy (MALDI-MS), Surface-enhanced laser desorp- tag DNA sequence, which is then quantiefi d from the tion/ionization mass spectroscopy (SELDI-MS), Bioaerosol supernatant by real-time PCR. These modifications enabled mass spectrometry (BAMS), Pyrolysis gas chromatography detection of RSV even at 4.1 PFU/mL. This assay oer ff s 8 Advances in Virology PCR amplicon ESI-MS Identification of virus Virus A Virus B Virus C Virus D (a) PCR-Electrospray Ionization/Mass Spectroscopy Silver film Silver nanorods Virus A Virus B Virus C Virus D Laser Spectra Silver vapor source Silver nanorods Oblique vapor desposition (b) Surface Enhanced Raman Spectroscopy Figure 3: A schematic representation of biophysical method of RSV detection. (a) PCR-electrospray ionization mass spectroscopy and (b) Surface enhanced Raman spectroscopy. a 4000-fold improvement in the limit of detection over ELISA possible using DNA hairpin structures functionalized to and a 4-fold improvement over detection when compared a gold filament, which is immersed in a capillary tube with real-time RT-PCR [59]. containing viral RNA and scanned for u fl orescence. eTh set- up wasabletodetectaslow as 11.9PFUs, whichwas∼200 2.6.2. Live Cell RNA Imaging. Live cell imaging can pro- times better than a standard comparison ELISA. vide the benefit of eeff ctive diagnosis and treatment, if the capability of identifying, monitoring, and quantifying 2.6.3. Quantum Dots. Immunou fl orescence microscopy biomolecules is developed. However, development of such based detection of RSV, that is, direct uo fl rescent-antibody systems for detection of viral agents is dicffi ult, especially in assay (DFA), is considered as gold standard [39], but the the early stages of infection. With the advent of molecular comparativeecffi acyofDFA with otherassaysdoesnot seem beacon technology, it is now possible to track mRNA of to have reached a consensus [39, 40, 94–97]. The prime host and RSV [32]. Based on this framework, a modifica- possible disadvantages and inconsistencies of DFA can be tion by oligonucleotide-functionalized gold nanoparticulate attributed to the fading of the dyes, conjugating antibodies probe was suggested as an improvement, wherein a gold with dyes, limited sensitivity due to background staining, and nanoparticle is functionalized with DNA hairpin structure by excitation at two different wavelengths [ 26–28]. To address thiol linkage. eTh DNA is so designed that the loop portion these issues, uo fl rescent nanoparticles, that is, quantum has a complimentary sequence to RNA to be detected and dots (QDs), appear as promising candidates for field clinical the 5 stem is linked to gold nanoparticle by thiol group and diagnostics. Due to their inorganic nature, they are less the 3 endislinkedtoafluorophore.Onhybridization to the susceptible to metabolic degradation. QDs are photostable; target RNA with loop, the uo fl rophore goes away from the that is, they do not lose u fl orescence on long exposure to light quenching gold and the emission is tracked. us, Th live imag- and can be excited at the same wavelength while radiating at ing of mRNA is possible. This mechanism of hairpin DNA different wavelengths and hence can be used for multiplexing functionalized gold nanoparticles (hAuNP) was executed by [27]. Various successful attempts were made to ameliorate Jayagopal et al. in detecting RSV mRNA in HEp-2 cells [33]. RSV detection using QDs. eTh progression of RSV infection This technique oer ff s the advantage of real-time detection in the HEp-2 cell line was studied using confocal microscopy of multiple mRNA at the same time, including the mRNA by QDs probing F and G proteins and it was found that of RSV and glyceraldehyde 3-phosphate dehydrogenase of this method was more sensitive than real-time, quantitative the HEp-2 cell. Quantitative assay using this approach was RT-PCR, particularly at early infection [29]. This approach Advances in Virology 9 was used by Tripp et al. in vitro,onVerocelllines and spectral signature [81]. Bacteria and viruses from various was extrapolated by an in vivo BALB/c mice study, which biological samples can be identiefi d, characterized, and clas- concluded the approach beyond diagnostics as it can be used sified from clinical samples [ 77, 82]. SERS can distinguish for multiplexed virus and/or host cell antigen detection and between DNA or RNA viruses like Adenovirus, Rhinovirus, intracellular tracking studies [28]. Rotavirus [83], and RSV [84]. Rotavirus is the common cause Flow cytometry is now widely used in diagnostics and the of gastroenteritis in children and the rapid and sensitive reliability is improved because millions of cells are analyzed detection was demonstrated with SERS [83]. SERS is an at a time and comprehensive data is produced [30]. Tracking established powerful tool for sensitive, expedited, and specific and targeting cellular proteins and the ease of multiple detection of various respiratory pathogens, like Mycoplasma parameters correlation allow flow cytometry to be used for pneumoniae and RSV. Using silver nanorod arrays (NA) various qualitative and quantitative assays. Flow cytometer platform for SERS, differentiation of M. pneumoniae in could detect RSV with sensitivity and reproducibility [31]. cultureand in spiked andtrueclinicalthroatswabsamples Agrawal et al. [26] showed that antibody conjugated QDs was achieved [82]. The notable sensitivity of SERS to resolve could detect RSV rapidly and sensitively using the principles strain level differences for RSV strains A/Long, A2, ΔG, and of microcapillary flow cytometry (integrated with a fixed- B1 can be exploited for clinical diagnosis instrumentation point confocal microscope) and single-molecule detection. [84]. A 40-nm carboxylate-modified uo fl rescent nanoparticles and streptavidin-coated QDs were used in their study, which 3. Prevention could estimate relative levels of surface protein expression. Prevention is the most important aspect of healthcare and 2.6.4. Gold Nanoparticle Facilitated Microarray. The FDA has prime contribution to the culmination of the disease approved microarray systems, semiautomated respiratory than the treatment measures. Effective preventive measures virus nucleic acid test (VRNAT), and the fully automated res- reduce the mortality and economic burden of the disease. piratory virus nucleic acid test SP (RVNATSP)(Nanosphere, Untilnow thereisnoeeff ctivevaccine forpreventionfor RSV. Northbrook, IL) are examples of the microarray based detec- Direct or indirect contact with the nasopharyngeal secretions tion systems for influenza A virus, influenza B virus, RSV or droplets (sneezing, coughing and kissing), fomites, and A, and RSV B [56, 98]. eTh se systems are based on the e-ffi food from infected patients can potentially transmit RSV. Live cient detection of microarray based hybridization using gold viruscansurviveonsurfacesforseveralhours[100],butatthe nanoparticles. The hybridization between the oligonucleotide cellular level, the viral spread is a series of systematic events probes and target DNA/RNA is detected specifically by of invasion including viral attachment and fusion followed hybridizing them again to gold nanoparticles functionalized by viral replication and protein synthesis. The F protein with oligonucleotide and the signal is generated by gold accumulates in the host membrane and then surrounds the facilitated reduction of silver in the presence of a reducing budding progeny viruses, thus spreading the infection to agent [99]. adjacent cells and exacerbating the infection (Figure 4). u Th s these proteins are considered as the potential candidate for 2.6.5. Surface Enhanced Raman Spectroscopy. Raman spec- the development of prevention measures such as antibodies, troscopy works on the basis of the inelastic scattering of DNA vaccines, and subunit vaccines. monochromatic radiation like near infrared, visible, or near ultraviolet, interacting with an analyte with low-frequency vibrational and/or rotational energy. But the signal generated 3.1. Antibodies. RSVhas threeenvelopeproteinsF,G,and is low and hence the signal is enhanced using silver or gold SH. Both F and G are glycosylated and represent the targets matrix substrates. er Th e are many modifications of Raman of neutralizing antibodies. eTh RSV F protein emerged as spectroscopy,but themostwidelyusedone is thesurface a good vaccine candidate due to its conserved and vital enhanced Raman spectroscopy (SERS) (Figure 3(b))[77]. role in cell attachment. Passive immunization is a direct Direct intrinsic, indirect intrinsic and extrinsic detection are approach to counter RSV (Figure 5). Initially, polyclonal three major SERS detection congfi urations [ 78]. In contrast antibodies from healthy human individuals resistant to RSV to IR spectroscopy,the Ramanspectra canbeobtained were successful in preventing RSV infection in high risk without the interference of water molecules and thus bio- infants and these pooled and purified immunoglobins were logical analytes can be studied in their native conformation. popular as RespiGam. The monoclonal antibody specifically SERS is routinely used for various bioanalytical purposes neutralizing F protein conferred eeff ctive protection against due to the rapid and nondestructive detection of analytes RSV as compared to RespiGam, and this licensed monoclonal with sensitivity, specificity, and precision even for a single antibody, palivizumab (Synagis), is now used to passively molecule or live cells [79, 80]. SERS can be targeted to protect high risk infants from severe disease, thus replacing various analytes that may constitute DNA, RNA, proteins, the RespiGam. The efficacy of the recombinant monoclonal or other organic compounds. Nucleic acids are the preferred antibody, palivizumab, has been tested for prophylaxis and candidates in biological SERS investigations, as the influence therapy in immunocompromised cotton rats [101]. Repeated of base composition and sequence, conformation (local doses of palivizumab were required to prevent rebound and/or global) of nucleic acids, or intermolecular dynamics RSV replication. Palivizumab is administered alone or in with protein or ligand is correspondingly expressed as typical combination with aerosolized ribavirin. Palivizumab cannot 10 Advances in Virology RSV Life cycle of RSV Budding RSV ready to infect adjacent cell Merging with the cell membrane Antigenome F protein Transcription Replication G protein Protein synthesis Assembly SH protein P protein N protein L protein Golgi complex Endoplasmic Nucleus reticulum Figure 4: A schematic representation of RSV life cycle. Modes of RSV inhibition G protein F protein + + No infection No binding to the cell RSV Gold nanoparticles Cell No infection + + No binding to the cell RSV Antibodies Drug No infection No binding Peptide to the cell RSV Fusion inhibitors Infection Fusion No treatment Binding RSV Figure 5: A schematic representation of various compounds inhibiting RSV binding to the cell. cure or treat serious RSV disease but neutralization of RSV palivizumab or motavizumab when pretreated with RSV, but can help in preventing serious RSV infections. Motavizumab there was a reduction in viral transcription, thus inhibiting has been found to neutralize RSV by binding the RSV both cell-cell and virus-cell fusion most likely by preventing fusion protein F aer ft attachment to the host, but before the the conformational changes in the F protein needed for viral viral transcription [102]. Viral entry was not inhibited by fusion. Advances in Virology 11 The effective use of palivizumab is limited due to the cost of RSV for IFN and immunopathology, a construct was and its use in infants with high risk of bronchiolitis based on designed by inserting the F gene into Newcastle disease the coverage by different healthcare systems [ 103]. In spite virus (NDV) vector (NDV-F). This modification served the of these restrictions on palivizumab, it has a wide societal purposeofhigherelicitation of IFNbyNDV-F than RSV, impact on use in infants with chronic lung disease due to protection against RSV infection without immunopathology premature birth or those with haemodynamically signicfi ant and enhanced adaptive immunity in BALB/c mice [109]. Wu cardiac disease. According to the modified recommendations et al. [110] developed a DNA vaccination strategy against of the Committee on Infectious Diseases of the Centers for RSV using a mucosal adjuvant. Two DNA vaccine vectors, Disease Control and Prevention of RSV, palivizumab is rec- namely, DRF-412 and DRF-412-P containing residues 412– ommended for infants with congenital heart disease (CHD), 524 of the RSV F gene, were cloned into the phCMV1 chronic lung disease (CLD), and birth before 32 weeks [104]. DNA vaccine vector. eTh DNA vaccine vectors DRF-412 Minimum 5 doses are recommended irrespective of the contained the cholera toxin gene region called ctxA2B acting month of the first dose for all geographical locations for as a mucosal adjuvant. eTh DNA vaccine was successfully infants with a gestational age of 32 weeks 0 days to 32 weeks expressed in mouse muscle tissue, which was conrfi med by 6 days without hemodynamically significant CHD or CLD. immunohistological analysis and RT-PCR. The immunized The new recommendations of the committee were aimed at mice induced neutralization antibody, systemic Ab (IgG, the high risk groups including infants attending child care IgG1, IgG2a, and IgG2b) responses, and mucosal antibody or one or more siblings or other children younger than 5 responses (Ig A) which mimicked the challenge with live RSV. years living with the child. Also, the infants were qualified The mice immunized with the DRF-412 vector contained less for receiving prophylaxis only until they reached 90 days of RSV RNA in lung tissue and induced a higher mixed Th1/Th2 age. Palivizumab, although eecti ff ve, is costly and thus is not cytokine immune response and had better protection than benecfi ial to the recipients especially during the periods when those immunized with the DRF-412-P vector, which was RSV is not circulating. A cost eeff ctive means of producing confirmed by lung immunohistology studies [ 110]. A rational RSV F neutralizing antibodies was experimented in phages approach to confer protection against various pathogens and plants. Much success in this regard of palivizumab through single vaccination or more often known as combined production was observed in the Nicotiana benthamiana plant or composite vaccines was employed for RSV, influenza A system which oeff red glycosylation and high production virus (INF-A), and herpes simplex virus type-1 [111]. Mice at lower upstream and equivalent downstream cost, when were immunized either by injection or by gene gun (gold compared to mammalian derived palivizumab. The efficacy of beads as carrier of the plasmid DNA) with mixture of four the plant derived palivizumab was more than the mammalian plasmids: INF-A haemagglutinin (HA), INF-A nucleoprotein derived palivizumab or the plant derived human monoclonal (NP), HSV-1 glycoprotein D (gD) and RSV glycoprotein F. antibodies in cotton rats [105]. This led to protection of mice from the respective pathogens of challenge; in addition, it also offered protection from 3.2. DNA Vaccines. RSV genome codes structural and func- Mycoplasma pulmonis challenge as well [111]. Recently several tional proteins that are immunogenic and referred for vaccine developments have been made in the eld fi of recombinant vaccines. In one such approach, Mycobacterium bovis Bacillus development against RSV. DNA based vaccines were devel- oped basedonthese proteins becausethe conceptwas simple, Calmette-Gue’rin (BCG) vaccine was modified to carry RSV as it involved a DNA fragment coding part or whole protein of NorM2and wastestedand foundtoestablish theTh1type immunity in RSV challenged mice [112]. The recombinant RSV that was inserted into an appropriate expression plasmid vector under a constitutive promoter control (Figure 6). vaccine also elicited the activation of RSV specific T cells eTh initial work with this approach was successful in the producing IFN-𝛾 andIL-2, alongwithreduction in weight expressionincells and in vivo murine models to eliminate loss, and lung viral protein load, thus establishing a Th1- the RSV infection, but the problem of RSV associated Th2 polarized immune response. Bactofection is bacteria mediated transfer of plasmid type immune response was persistent. This problem was attempted to be resolved by manipulating the parameters DNA into mammalian cells. Xie et al. employed this interest- of choice of the protein to be expressed, the expression ing way of delivering and expressing the DNA vaccine in mice against RSV [113]. This approach serves the purpose of natu- vector, adjuvants, formulations, and intracellular stability of the plasmid. rally activating the immunostimulatory response of the host Mice challenged with the RSV-G construct had balanced andalsodeliveryofthe DNAvaccine.Attenuated Salmonella systemic and pulmonary Th1/Th2 cytokines and RSV neutral- typhimurium strain SL7207 expressing vector pcDNA3.1/F izing antibody responses [106]. However, the RSV F protein containing the RSV F gene was orally administered to BALB/c gene is considered as a widely used and prospective target mice, triggering efficient antigen-specific humoral, cellular, for the development of vaccines and is oen ft a favourite and mucosal immunity [113, 114]. model for DNA vaccines against RSV [107]. But the wild A novel method of developing RSV DNA vaccine was devised by replacing the structural genes with RSV genes type RSV F protein expressed from DNA plasmid was poorly expressed, so a codon optimized DNA vaccine was in an attenuated strain of Venezuelan equine encephalitis designed for better in vivo expression and hence was more virus (VEEV). VEEV has an ssRNA (+) genome and con- tains a strong subgenomic promoter. eTh replicon particles immunogenic and reduced the RSV titer [108]. To address the low immunogenicity of F protein, the antagonistic activity were prepared by providing helper RNAs encoding VEEV 12 Advances in Virology DNA functionalized DNA encapsulated DNA vaccine nanoparticles nanoparticles Viral DNA Naked DNA vector Expression vector RSV Vector Mice protected Vaccination RSV challenge RSV No vaccination RSV challenge RSV infection Figure 6: A schematic representation of a simple DNA vaccine administered as a naked DNA vector or functionalized nanoparticle or as an encapsulation for controlled delivery. capsid andenvelopeglycoproteinwhich comprise structural virus like particles (VLP) developed from NP and M proteins proteins, and all of these, when transfected into Vero cells, of Newcastle disease virus (NDV) and a chimeric protein resulted into a replicon particle that could independently consisting of cytoplasmic and transmembrane domains of synthesize the RSV protein, thereby activating the immune NDV HN protein and ectodomain of the RSV G protein response and protection. eTh system could be modulated by (H/G) [122]. These VLPs resulted in an immune response the administration of helper RNAs. This strategy was applied better than UV-inactivated RSV and provided complete to the mice and rhesus monkey models, conferring protection protection in mice from RSV even at a single dose, with against RSV and a desirable extent of a balanced Th1/Th2 type elicited neutralizing antibodies. eTh VLP-H/G-immunized immune response was received [115]. mice did not show enhanced pathology as compared to FI-RSV.Inanother approach to solvethe immunogenicity relatedproblemsindevisinganRSV vaccine,arecombinant 3.3. Subunit Vaccines. Several approaches have been con- sidered for developing an eeff ctive vaccine against RSV vector, based on either murine PIV type 1 or Sendai virus was used to deliver RSV G protein through reverse genetics [123]. including the formalin inactivated RSV vaccine developed in This provided effective protection against RSV in cotton late 1960s, which instead resulted in an enhanced infection [116]. An efficient RSV vaccine would be the one with proper rats. Another similar study using Sendai virus (SeV) as a vaccine carrying RSV F gene provided protection against four balance between immunogenicity and protection without any pathogens including hPIV-1, mouse PIV-1, hPIV-3, and RSV allergic response [8]. RSV F has widely been accepted as the vaccine candidate [124]. This approach has been used to compare two vaccine models, one with SeV backbone and the other with PIV-3 due to its conserved nature among various strains as well as among the other paramyxoviruses [8, 117–119]. An antigenic backbone. eTh SeV based vaccine showed a decrease in RSV load in African green monkeys lung titers. region corresponding to RSV F protein (region 255–278) Another study with recombinant vaccine utilizes recom- was cloned into a vector with ctxA2 B gene of cholera toxin and named as rF255, which elicited a helper T cell binant simian Varicella viruses (rSVVs) which express RSV G and M2 protein genes and was evaluated in the Vero type 1 immune response in mice. It also resulted in higher cell line [125]. Such a recombinant vaccine approach could expression of serum neutralizing antibody in immunized mice [120]. Similarly, a multivalent recombinant protein was be very useful in elderly people with risk of infection with Varicella andRSV,aswellaschildrenwithchickenpoxand developed by cloning RSV F, M2, and G protein into a bacterial pET32a (+) vector (called rFM2G), which resulted RSV. rSVV based vaccines resulted in enhanced immune responses to RSV antigens serving as suitable vaccines in in enhanced serum IgG titers [121]. rFM2G was also used rhesus monkeys. A very similar approach was adopted for in conjunctionwithflagellinasanadjuvantwhich didnot increase the IgG titers. In an eo ff rt to address the issues of developing a recombinant alphavirus or immune stimu- lating complex (ISCOM) antigen against RSV [126]. The immunogenicity, several studies have used the approach of recombinant vector was designed by using a self-abortive combining other viruses with RSV genes. One such study uses Advances in Virology 13 alphavirus called Semiliki forest virus carrying RSV F and and found to provide significant protection against RSV, G genes. eTh advantages of using such recombinant abortive along with RSV specicfi humoral responses and an enhanced viruses are that they can undergo viral infection just once, Th1/Th2 response [ 131]. The NE rendered the RSV ineffective eliminating chances of adverse eeff cts resulting from further when treatedfor2or3hours, with areduction in viraltiter infection, and infecting the host cell resulting in expression up to 10-fold as compared to media controls. NE-RSV also led of the inserted RSV gene followed by humoral and cell to higher expression of RSV specific antibody, with significant mediated immune response. eTh ISCOM used was composed decrease in viral load, no hyperreactivity, and no Th2 cytokine of Quillaja saponin, lipids, and RSV antigens, having adjuvant induction. u Th s, this novel approach seems to be a promising properties as well. ISCOM has been shown to enhance and safe vaccine option against RSV. antibody production, T cell proliferation, and MHC class- The bitter episode of formalin inactivated RSV vaccine I responses as well as RSV specific neutralizing antibodies, has impeded the vaccine development and in fact has raised IgG and IgA. The recombinant vaccine SFV/F or SFV/FG serious concern in the use of native RSV or its components. resulted in IFN gamma response along with resistance to This approach was revived with a novelty, wherein the RSV infection without worsened RSV disease, whereas the engineered RSV F protein aggregates formed nanoparticles resultswereoppositeinISCOM/FGwithenhancedgobletcell and were used as vaccine, and these nanoparticles induced hyperplasia post-RSV challenge. protective immunity in cotton rats [132]. To combat RSV, host neutralizing antibodies are always more preferred than the 3.4. Nanovaccines. Recently, several applications of nan- therapeutic antibodies. But the epitopes of the neutralizing otechnologyhaveappearedinthe developmentofvaccines antibodies are larger than those of the therapeutic neu- tralizing antibodies (palivizumab and motavizumab). us, Th popularly known as Nanovaccines. DNA vaccine is prone to rapid degradation when introduced into an animal system; the use of these epitopes for neutralizing antibodies as a so to increase the retention and increase the efficacy of the vaccine requires the retention of immuno functional con- DNA vaccines, they can be encapsulated into a polymer formation while getting rid of the undesired protein. RSV F that will protect and facilitate controlled release (Figure 5). oligomeric protein nanoparticle was synthesized by inserting recombinant RSV F gene into Baculovirus and expressed Various synthetic or natural polymers are now experimented for targeted delivery and controlled release of the carrier. in Sf9 insect cell lines. This resulted in high recombinant Chitosan is the polymer of great interest in respiratory protein expression compared to native protein. This also resulted in the conformation of rosette nanoparticles which disease treatment, because of its mucoadhesive property and biodegradability, which balances the purpose of longer reten- wasthe aggregateofmultipleRSV Foligomers foundtobe tion and controlled release of carrier molecules encapsulated. immunogenic. The study was extrapolated to phase 1 human u Th s, chitosan nanoparticles are being developed against clinical trials, which showed its safety and efficacy against RSV. A DNA vaccine (DR-FM2G) constitutive expression RSV [133]. vector consisting of antigenic regions of RSV F, M2, and G genes driven by human cytomegalovirus promoter was 3.5. Nutrition. Nutrition is not considered as treatment encapsulated using chitosan nanoparticles (DCNPs). The but is a prerequisite for homeostasis, and any nutritional advantages of DCNPs are that it was more stable than naked imbalance attracts disorders and diseases. Supplementary DNA or chitosan and so ideal for protection against DNA nutrients could reverse the adverse eeff cts of the disorders degradation by nucleases. The sustainability of the DCNPs in and diseases, so in sensu nutrition can serve both as treatment mice was more than naked DNA which was correspondingly and prevention. Carbohydrate rich diets and diet lacking indicated by higher RSV protein expression evident by antioxidants like fruits and vegetables during pregnancy are immunohistochemical and real-time PCR studies [127, 128]. suspected for RSV susceptibility [134]. Resveratrol (trans- A similar study was conducted by Eroglu et al. [129], where 3,4,5-trihydroxystilbene), a polyphenol from grapes has thehighlyconserved RSVFgene wasclonedintopHCMV1 demonstrated reduction in RSV replication and inflamma- expression vector and encapsulated into poly-hydroxyethyl- tion [135, 136]. A study demonstrated that the cord blood methacrylate nanospheres coated by chitosan and transfected deficient in vitamin D was associated with RSV bronchiolitis; into Cos-7 cells. eTh transfection efficiency of the system the neonates were at higher risk of RSV in the rfi st year was at par with commercially available transfecting agents. In of life [137]. Vitamin D inhibits NF-𝜅 B signalling which vivo studies with the BALB/C mice also indicated F protein is responsible for RSV inflammation without affecting the expression and reduced RSV infection. antiviralactivityofthe host [138]. Lower levels of micronutri- Avaccine basedonthe recombinantRSV Ngenerings, ents like zinc, copper, selenium and retinol (Vitamin A), and called N-SRS, enclosing a bacterial RNA has been developed alpha-tocopherol (Vitamin E) were observed in the children and assessed intranasally in BALB/c mice [130]. N-SRS was aeff cted by RSV and human metapneumovirus [ 139]. In one adjuvanted with E. coli enterotoxin LT (R192G) with efficient study, RSV infected children were administered vitamin A protection against RSV and high titers of IgG1, IgG2a and to compensate the lower vitamin A serum level which was IgA, andsoforth.Althoughthisnanovaccine elicited amild found benecfi ial for children with severe RSV infection [ 140]. inflammatory response in airways of the mice, it enhanced Also, probiotic diet has shown to boost resistance against + + the expression of antigen specific CD4 and CD8 responses. pathogen by modulating immune response, as in case of In another novel approach, nanoemulsion (NE) was used RSV, Lactobacillus rhamnosus (a probiotic bacteria) treated as an adjuvant to mucosal RSV vaccine in a mouse model BALB/c mice showed significantly reduced lung viral loads 14 Advances in Virology and pathology after the RSV challenge [ 141]. These studies shock protein (HSP90) for its replication [165]. The anti- indicate the importance and association of nutrition with RSV activity of these compounds was seen in human airway RSV susceptibility. epithelial cells (HAEC) and is considered as drug resistant therapeutics, due to the highly conserved target chaperon protein. eTh se compounds are also known to inhibit repli- 4. Treatment cation of HPIV, influenza virus, and rhinovirus. eTh anti- There are very limited treatment options available for RSV. viral activity dose is not toxic to the cells and inhalation However, there are many drugs for the symptoms associated mode of treatment can increase the local efficacy and avoid with RSV infection. eTh target genes and proteins vital for unnecessary exposure to other organs [166]. RSV infection (discussed in Section 3)are importantfor developing preventative and treatment measures. eTh mode 4.2. Fusion Inhibitors. Recent advances in the development of action and potency of a drug determines the approach of anti-viral drugs include the fusion inhibitors. eTh fusion of prophylactic or curative application. Considering the inhibitors are usually synthetic compounds or molecules proposed life cycle of RSV, theoretically, there are numerous interrupting the fusion of virus with the host cell usually by modes to interfere with RSV infection, but these options may binding the fusion proteins (Figure 5). The fusion inhibitors not be practical. Replication, transcription and fusion are the have been widely studied as anti-viral agents in several viruses few target processes for drug development against RSV. A including HIV, RSV, Henipavirus, Hendra virus, Nipah virus, focus is therefore on development of potent drug which holds Paramyxovirus, metapneumoviruses, HIV, and RSV [11, 167– conformity in the human trials. Some of the approaches are 173]. The first reports of the use of peptide(s) as fusion described below that promise to be a potential treatment for inhibitors include the development of DP-178, a synthetic RSV (Table 2). peptide based on the leucine zipper region of the HIV fusion glycoprotein gp41 [167], which showed an IC50 at 0.38 nM 4.1. Antiviral Drugs. Ribavirin or 1-[(2R, 3R, 4S, 5R)-3,4- against HIV-1. Fusion inhibitors for the paramyxoviruses dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-tri- have also been developed based on the conserved region azole-3-carboxamide is a widely used broad spectrum of the fusion protein F. The F protein is widely known for synthetic anti-viral drug for both DNA and RNA virus its conserved nature among the Paramyxoviridae family [7]. treatment. Oral and nasal administration of ribavirin Lambert et al. [174] developed the fusion inhibitors belonging for treatment of severe lower respiratory tract infections to the conserved heptad repeat (HR) domains of F1 region caused by RSV and influenza virus are an option [ 104]. of F protein which is analogous to the peptides DP-107 and Ribavirin is phosphorylated in the cells and competes with DP-178 of HIV gp41. These fusion inhibitors were tested 󸀠 󸀠 adenosine-5 -triphosphate and guanosine-5 -triphosphate against RSV, human parainfluenza virus 3, and measles virus, for viral RNA-dependent RNA polymerases in RNA which showed antiviral activity specific to the species of viruses. However, the mechanism of ribavirin differs for origin. DP-178 is an FDA approved anti-HIV drug with DNA viruses, as it is a competitive inhibitor of inosine International Nonproprietary Name (INN) Enfuvirtide and monophosphate dehydrogenase (IMPDH), causing deletion trade name Fuzeon. Out of the peptides tested, the peptide of GTP and messenger RNA (mRNA) guanylyl transferase T-118 developed from RSV was the most eeff ctive, with an (mRNA capping enzyme) and adversely affecting protein EC50 of 0.050𝜇 M. These fusion inhibitors were then further synthesis [142, 143]. The exact mechanism of anti-viral characterized and tested. It was shown that dieff rent fusion activity of ribavirin against RNA and DNA viruses is still inhibitors derived from same HR region differ in their anti- not clear. eTh usefulness of ribavirin against viruses is viral activity [147]. The HR121 and HR212 peptides showed an not only due to its anti-viral activity but also due to its IC50 of 3.3 and 7.95𝜇 M, respectively, against RSV. Similarly, capability to modulate the immune system. Ribavirin is another peptide inhibitor was developed from Rho-A which suggested to have immuno-stimulatory eeff cts on Th cells showed inhibition of syncytia formation induced by RSV [144]. The derivatives of ribavirin such as viramidine, [148]. RhoA (a small GTPase) is involved in many biological merimepodib, and other IMPDH inhibitory molecules processes and was shown to bind the RSV-F protein at amino like mycophenolate and mizoribine have shown antiviral acids 146–155. A peptide derived from the RSV F binding activity against the hepatitis C virus, and hence, there is domain of RhoA (RhoA77-95) was shown to inhibit RSV and scope for investigating them as potential anti-RSV drugs PIV-3 infection and syncytium formation, block cell-to-cell [143, 145]. eTh re are many other compounds that can inhibit fusion, and reduce viral titers and illness in mice. RSV replication and a well-known compound RSV 604 The fusion inhibitors are not only limited to peptide ((S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H- inhibitors; but a range of chemical inhibitors have also been benzo[e][1,4]diazepin-3-yl)-urea) showed promising results tested against RSV and benzimidazoles are well-known against RSV [146]. fusion inhibitors [175]. A lead compound was identified A derivative of antibiotic geldanamycin 17-ally-lamino- to subsequently synthesize an analogue JNJ2408068, a low 17-demethoxygeldanamycin (17AAG) and 17-dimethylam- molecular weight benzimidazole, which showed high anti- inoethylamino-17-demethoxygeldanamycin (17DMAG) tar- viral activity. It had an EC50 of 0.16 nM, 100,000 times better geted against cancer has now attracted researchers due to its than that of ribavirin. This compound showed anti-fusion antiviral property. These compounds are HSP90 inhibitors activity against RSV in a dual mode of action including and thus helpful against RSV, as RSV is dependent on heat prevention of cell-virus fusion activity as well as cell-cell Advances in Virology 15 ff ff fl Table 2: Comparison of different treatment approaches for RSV. Treatment Mechanism Example Remark References Replication inhibition RSV 604 Eective against RSV, but adverse effect on [104] Mutation Ribavirin, viramidine, merimepodib Antiviral drugs Inhibitor of inosine monophosphate the host [142–146] Ribavirin, mycophenolate, mizoribine dehydrogenase Immunostimulatory eects Ribavirin Peptide fusion inhibitors promising anti Peptide—HR121, HR212, RhoA Fusion inhibitors Inhibiting fusion protein attachment to cell RSV drug; chemical fusion inhibitors have [147–150] Chemical—BMS-433771, RFI-641 side effects Nanoparticles Inhibiting attachment to cell Silver nanoparticles, gold nanoparticles Emerging field, conclusive studies required [ 151, 152] siRNA-ALN-RSV01 Effective and safe; ALN-RSV01 completed Antisense therapy RNA interference [153–157] Phosphorodiamidate morpholino oligomers phase IIb clinical trails Plant extracts—Cinnamomum cassia, Cimicifuga foetida, Probably fusion inhibitors, Ethnobotanicals Sheng-Ma-Ge-Gen-Tang, Ginger, etc. Promising but conclusive studies required [158–164] anti-inammatory Decoctions-modified Dingchuan, Liu-He-Tang, water extract of Licorice 16 Advances in Virology fusionactivity.However,itwasfoundineeff ctiveagainstother eeff ctive against 6 laboratory and 18 clinical viruses at con- viruses of the family including HPIV-3 and measles virus. centrations between 0.008 and 0.11 mM (0.013–0.18 mg/mL). A vast screening of as many as 16,671 compounds (source The compound reduced the viral load to an extent of 1.7 logs ChemBioNet library) was conducted for anti-RSV activity in theAfrican greenmonkeymodel andalsoinmiceand in vitro and two novel compounds, N-(2-hydroxyethyl)-4- cotton rats. In order to further enhance the antiviral activity methoxy-N-methyl-3-(6-methyl[1,2,4]triazolo[3,4-a]phthal- of RFI-641, it was modified by replacing its triazine linkers azin-3-yl)benzenesulfonamide (named as P13) and the with pyrimidine [182]. However, this modification did not 1,4-bis(3-methyl-4-pyridinyl)-1,4-diazepane (named as C15) have much difference in the anti-viral activity, thus rendering were mined, which reduced the virus infectivity with IC50 this modification not much of practical use. er Th e are several values of 0.11 and 0.13𝜇 M, respectively [176]. novel nonbenzimidazole based compounds, showing anti- Recently, several groups have reported synthetic fusion RSV activity in vitro, but a more polar compound thiazole- inhibitors of RSV, especially the benzotriazole derivatives imidazole 13 was selected on the compound potency, mod- [177]. Aeft r evaluating the structure-activity relationship erate permeability, and low metabolic rate in rats, and more (SAR) of these compounds, named as series 1 compounds, detailed in vivo studies are further anticipated [183]. it wasobservedthatthe topology of thesidechainsofthese compounds is important and facilitates the modicfi ation 4.3. Nanoparticles. It has been established that metals like of their physical properties, as many of these compounds silver [184] and gold [185]haveanti-microbialactivity, but showed poor therapeutic indices (cytotoxic effects) to the cytotoxic effects of these reactive metals make them unsuit- host cells tested. In order to address these issues, a second able for their use in humans. The reactivity and behaviour of series of derivatives of the compound 1 were developed and metals canbemodulated by reducing theirsizetonanoscale. evaluated for SAR and their functionality as fusion inhibitors Carbon nanotubes (CNTs) are emerging nanomaterials for [178]. These compounds were developed from 1,which had biomedical application [186]. Polyvinylpyrrolidone (PVP) a tolerant diethylaminoethyl side chain with both polar and conjugated silver nanoparticles showed low toxicity to HEp- nonpolar functionality against RSV and had a replacement 2 cells at low concentrations and exhibited 44% RSV inhi- of the benzotriazole with benzimidazole-2-one. These were bition [151](Figure 6). Singh et al. used fusion inhibitor potent inhibitors of RSV in vitro.Thesecompounds were peptide functionalized gold nanoparticles and carboxylated named 2 and had an additional structural vector absent gold nanoparticles of size 13 nm against RSV, which showed in 1, which accounted for enhanced potency as fusion 83% and 88% inhibition of RSV, respectively [152]. Similar inhibitors and served as the base for further development approach wasemployedbyrecombinant RSVFprotein of fusion inhibitors. Further, these group 2 compounds were functionalized on gold nanorods [187]. The emergence of modified by introducing acidic and basic functional groups nanotechnology has opened new avenues for RSV treatment. into the side chains [179]. The oxadiazolone had anti-RSV activity comparable to that of ribavirin, whereas the ester modified group 2 compounds were suitable for oral admin- 4.4. Antisense Treatment. RNA interference (RNAi) which istration. These studies further led to the identification of is a normal cellular event has become a powerful means of a benzimidazole-2-one derivative called BMS-433771 which controlling gene regulation. The interference mediated by was an orally active RSV inhibitor [149]. Another compound siRNA was used against human immunodeficiency virus, studied was the 5-aminomethyl analogue 10aa with potent poliovirus, hepatitis C, and parainu fl enza virus (PIV) in cell anti-RSV activity towards BMS-433771 resistant RSV. eTh culture [188–190]. The concept of inhibiting RSV infection compound BMS-433771 was further modified at side chains using targeted antisense mechanism was applied by Jairath and with the introduction of an aminomethyl substituent et al. by silencing the RSV-NS2 gene [191]. Following the at the 5-position of the core benzimidazole moiety [180]. RNAi approach, Bitko et al. designed siRNA against the P The aminomethyl substitution in the benzimidazole ring was gene of RSV and PIV which protected mice against individual found to enhance the antiviral activity. and mixed infections upon intranasal administration [192]. Furthermore, the consecutive modicfi ation of benzimi- The effectiveness of siRNA action was observed with and dazole resulted in benzimidazole-isatin oximes which were without the use of transfection reagents. This approach was evaluated for anti-RSV activity [181]. The compound was ana- also effective when targeting the RSV-F gene [ 193]. Similar lyzed for its antiviral activity, cell permeability, and metabolic work on HEp-2 cell lines was replicated using four siRNA, stability in human live microsomes. Several other derivatives designed to silence RSV F gene, which showed inhibitory with modification such as O-alkylation and addition of action against RSV at various concentrations [194]. Silencing nitrogen atoms to isatin phenyl ring were implemented also different RSV genes too had an inhibitory action on the to enhance antiviral activity. Three compounds 18j, 18i,and RSV, a plasmid encoding siRNA which was complexed with 18n were shown to have anti-viral activity against RSV in the chitosan targeting RSV-NS1 gene decreased RSV infection BALB/c mice. Further, a compound RFI-641 was identified in BALB/c mice and Fischer 344 rats and also reduced which was found to be the most potent anti-RSV agent theassociatedinflammation [ 195, 196]. Zhang et al. showed inhibiting RSV both in vitro and in vivo andisinphase I that siRNA nanoparticle targeting RSV NS1 gene resulted in clinical trials. RFI-641 is a biphenyl triazine synthesized by increased IFN-𝛽 and IFN-inducible genes in A549 cells and coupling diaminobiphenyl to two chlorotriazine molecules in human dendritic cells, elevated type-1 IFN, and increased under microwave conditions [150]. RFI-641 was found to be differentiation of CD4 Tcells to Th1 cells[ 196]. Also Advances in Virology 17 mice treated with siNS1 nanoparticles exhibited significant Particularly in neonates, RSV bronchiolitis increases IL- decrease in lung viral titers and inflammation. 4𝛼 levels which results in increased Th2 response, so an An interferon-inducible enzyme, 2-5A-dependent RNase, antisense oligomer was synthesised for the local silencing present in higher vertebrates requires 5 -phosphorylated, of the IL-4𝛼 gene. Intranasal application of the antisense 󸀠 󸀠 2 ,5 -linked oligoadenylate (2-5A) for its endoribonuclease oligomers into a neonatal murine model reduced the Th2 activity against single-stranded RNAs. This feature of 2- type mediated pulmonary pathological signs of inflammation 5A-dependent RNase is looked upon as an effective RSV and lung dysfunction [203]. A combinatorial approach of the treatment [197]. RSV upon infection elicits immune response anti-sense oligomer against RSV and IL-4𝛼 would control of the host and particularly the interferon levels [68], and RSV infection and the adverse effects of RSV mediated this phenomenon is exploited for the anti-RSV activity of inflammation. 2-5A-dependent RNaseL. The endoribonuclease activity of Phosphorodiamidate morpholino oligomers (PMOs) are 2-5A-dependent RNaseL was used for targeting RSV M2 the oligomers where the nucleobases are covalently attached 󸀠 󸀠 gene specicfi allybycovalent2 –5 oligoadenylate target with the morpholine ring replacing the deoxyribose sugar antisense, which resulted in the reduction of RSV replication. while the phosphodiester bond is replaced by the phosphoro- The endoribonuclease activity had negligible eeff ct with an diamidate linkage [157]. Morpholino chemical modification inactive dimeric form of 2-5A linked to antisense, 2-5A linked of RNA can be used as antisense with the advantages of to a randomized sequence of nucleotides, and antisense specificity, in vivo stability, and targeted delivery. PMOs block molecules lacking 2-5A and did not affect the other RSVs the target complementary RNA and the target RNA fails or cellular RNAs [198]. Additionally, to widen the range of to interact with the proteins and thus the RNA function the approach, the effects of modicfi ation of oligonucleotides is hindered. This is specifically true for mRNA and its and RNA target sites were studied [199]. This model was translation. This phenomenon is different from antisense as improved with respect to the specificity and activity by the RNase H is not involved [204]. Hence PMOs can be a chimera of 2-5A-antisense, christened as NIH351. The designed containing the initiation codon against viruses to sequence information of RSV genome was used to develop be anti-viral. Better intake into the cell and in vivo systems NIH351 and was 50- to 90-fold more potent against RSV can be facilitated by conjugating cell penetrating peptide(s) strain A2 than ribavirin [200]. Administration of siRNA in (arginine-rich peptide (RXR) 4XB) to it. This approach was combination with ribavirin was recommended for eeff ctive attempted against the RSV-L gene to inhibit RSV in cellular treatment [201]. The parent molecule was chemically modi- and murine models [205]. fied to further increase the in vivo stability and specicfi ity and potency of NIH351. The resulting new version RBI034 was 4.5. Ethnobotanicals. Various natural and synthetic chemical ∼50% more eeff ctive than the parent molecule against RSV compounds have been screened for their application to (strain A and B) and was not cytotoxic in the effective dose treat RSV infections. Plants are rich sources of alkaloids, ranges. RBI034 treatment of African green monkeys shows steroids, flavonoids, and other complex compounds that have promising results [202]. medicinal value and medicinal plants contribute significantly Alvarez and coworkers [153]cameupwithanewRSV- to the traditional Indian and Chinese medicine. Ancient NS1 gene specific siRNA (ALN-RSV01) having a broad Chinese literature has descriptions of plant extract against spectrum of antiviral activity that targeted the nucleocapsid respiratory diseases [206]. The exact active compound or gene of RSV. In vivo BALB/c murine studies demonstrated action of the traditional formulations is not understood that intranasal dosing of ALN-RSV01 resulted in a 2.5- to [207]. Now, modern assays have made it possible to get an 3.0-log-unit reduction in RSV lung concentration. To scale insight into the mechanism and purification of active plant up this molecule for RSV treatment in humans, the safety, based compound. Extracts of Lonicera deflexicalyx (Chin tolerability, and pharmacokinetics were tested on healthy Jinyinhua) were tested against RSV. The active compound adults, demonstrating its safety and tolerance in human from the extract was 3, 5-dicaeff oylquinic acid (CJ 4-16- subjects [154]. In the human clinical trials of ALN-RSV01, 4), which was isolated, and purified by a series of chro- healthysubjectsweregrouped andadministeredeithera matographic processes. It is suggested that CJ 4-16-4 is a placeboorALN-RSV01 nasalspray forRSV.Therewas 44% fusion inhibitor and the in vitro and in vivo studies suggest reduction in the RSV infection in the subjects who received that it is a more eeff ctive RSV inhibitor than ribavirin ALN-RSV01 without any adverse effect. u Th s, this study in [208]. Cytopathic effect (CPE) assay based screening showed real terms has established a unique “proof-of-concept” for an that 27 of 44 herbs had moderate or potent anti-RSV RNAi therapeutic agent in RSV treatment [155]. ALN-RSV01 activity [206]. The plant extracts from Cinnamomum cas- proved to be safe and was eecti ff ve against RSV even in a sia, Cimicifuga foetida, Sheng-Ma-Ge-Gen-Tang (SMGGT) complex clinical situation like lung transplants, which was a (Shoma-kakkon-to), Xiao-Qing-Long-Tang (Sho-seiryu-to, remarkable achievement [156]. It is the product of Alnylam so-cheong-ryong-tang), Ge-Gen-Tang, and Ginger (Zingiber Pharmaceuticals, Inc. and has completed phase IIb clinical ocina ffi le) show anti-RSV activity. These extracts probably trials. inhibit RSV infection by blocking the F protein binding to the Another approach of RNAi treatment to combat RSV cell and some of the extracts even stimulate IFN-𝛽 production is to decelerate the adverse effects of RSV mediated [158–163]. Some decoctions like modified Dingchuan (con- Th2 type immune response because the aggravated host sists of Salviae miltiorrhizae radix, Scutellariae radix, Farfarae immune response is more harmful than RSV infection itself. flos and Ephedrae herba), Liu-He-Tang (consist of 13 plant 18 Advances in Virology extract), and water extracts of Licorice (Radix glycyrrhizae of the respiratory viruses [6, 213]. The situation is more and Radix glycyrrhizae Preparata) have shown eeff ctiveness intricate as some authors report that coinfection with non- against RSV in vitro. Moreover, the modified Dingchuan RSV respiratory viruses tends to increase RSV severity [214] decoction (MDD) exhibited anti-inflammatory and anti-viral and also there is a hypothesis of a synergistic association of eect ff in mice (SPF ICR mice) infected with RSV. MDD RSV with other viral or bacterial infections [215]. u Th s, this suppressed eotaxin, IL-4 and IFN-𝛾 level in serum, and topic is a subject of debate and these contradictions need mRNA expression of TLR4 and NF-𝜅 B in lungs of RSV claricfi ation and consensus for proper treatment options. A infected mice [164]. link between atopy, asthma, and RSV was suspected for a very long time, but now there is supporting evidence for this possible relationship [216–218]and is also ascribed partly to 5. Challenges in the Diagnosis, genetic factors of RSV and the host [70, 71, 219, 220]. The Prevention, and Treatment of RSV activity of RSV in the community may be aeff cted by many Technology has provided enough capabilities for the detec- factors, including climate, air pollution [221], race/ethnicity tion of RSV in various sample types at various stages of [222], and social behavior of the population. Under these infection using an array of techniques, but the challenge is, complex factors associated with RSV, it is established that availability of these facilities at the correct time for a reason- early detection of risk factors and medical intervention can reduce the incidence of RSV [223]. A balance of execution able cost. eTh most important perspective of RSV diagnosis is the strategic management of choice between the point and/orabeyanceofprophylacticmeasuresiscriticalwith of care testing and central laboratory testing [36]. Though respect to the above discussed determinants [224]. There is limited data to correlate RSV global transmission dynamics the point of care testing gives rapid detection advantage, it sueff rs from lower sensitivity and thus is a problem to with climate and population [2], due to which it is difficult to be dealt with. Rapid diagnostic tools like RT-PCR/ESI-MS, develop strategies for RSV prevention and treatment. microarray based semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus 6. Conclusions nucleic acid test SP (RVNATSP) (Nanosphere, Northbrook, IL) have proved their efficiency, but their application in Currently, there is no vaccine or effective treatment against routine clinical practice is still a challenge. RSV, but the rapid and sensitive RSV detection is possible. There are numerous molecules that can be potential eTh detection techniques are ameliorated by incorporating antiviraldrugs,but thescreening of avastnumberof one or more methods and with the advancement in material compounds is cumbersome; hence, high throughput filtering science and biophysical capabilities, it has reinforced the is an essential part of drug development. As an example, development and design of RSV detection systems. However, when 313,816 compounds from the Molecular Libraries Small an eecti ff ve detection technique can be transformed into Molecule Repository were screened against RSV in HEp-2 effective diagnosis by integrating it into the community cell line, only 409 compounds showed 50% inhibition of the health monitoring program at a reasonable cost. Prevention cytopathic effects [ 209]. The challenge after this sophisticated of RSV infection at present is limited to only high risk screening is the translation into drugs by clearing the phases individuals with a limited ecffi acy. New preventive measures of in vivo animal studies and human trials. A setback for research like DNA vaccines, subunit vaccines, and nano- the development of therapies against RSV is the lack of a vaccineshavereached animal trials.Onthe otherhand, good animal model as they do not truly manifest effects of the RSV treatment approaches using antisense oligomers, RSV infection as in humans. RSV experiments in various fusion inhibitors, and benzimidazole drug have proceeded animal models like BALB/c mice, cotton rats, macaques, into clinical trials. eTh challenges associated with RSV African green monkeys, owl monkeys, cebus monkeys, bon- management are categorically numerous. However, at the net monkeys, olive baboons, and chimpanzees are evident current pace of scienticfi research and development and with in the literature. Small animal models like BALB/c mice the implementation of scientific, commercial, and program andcottonratsare commonly used duetoeaseofhandling recommendations to develop epidemiological strategies, it and low cost, whereas the primate studies are conducted seemsoptimistictohaveaneeff ctivediagnosis,prevention, with more stringent regulations and bear heavy expenses and treatment solution for RSV in near future. [210, 211]. eTh re are many aspects that need to be addressed in the challenges for vaccine development programs and Competing Interests the technological interventions to deal with RSV [212]. These challenges include safety issues concerning the subjects eTh authors declare that they have no competing interests. involved in clinical trials, as evident by the failure of formalin inactivated RSV vaccine and motavizumab at the clinical trial levels which resulted in undesired immunogenic responses in Acknowledgments the patients involved [34, 116]. Though the present data conclude that RSV is one of the The authors acknowledge the National Science Foundation leading causes of morbidity and mortality in children and Grant NSF-CREST (HRD-1241701) and HBCU-UP (HRD- elders, there is no significant correlation between increased 1135863). They thank Eva Dennis for the gur fi es used in this disease severity, respiratory deaths, and detection of any paper. 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