Poliovirus nonpermissive CD155 knockout cells derived from RD cell line for handling poliovirus potentially infectious materials in virology laboratories.

Destruction of all poliovirus containing materials, safe and secure handling of retained polioviruses for vaccine production, and research will be obligatory to eliminate facility-associated risks. Polioviruses and poliovirus potentially infectious materials (PIM) including fecal or respiratory samples requiring containment have been defined in World Health Organization-Global Action Plan (GAP III) documents. Non-polio laboratories culturing viruses from PIM are most affected as cell cultures of human and monkey origin are also poliovirus permissive. CRISPR gene-editing technology was used to knockout the poliovirus receptor (PVR/CD155) gene in the rhabdomyosarcoma (RD) cell line. PVR knockout RD cell susceptibility was tested using known non-polio enterovirus (NPEV) types. A selected clone (RD-SJ40) was field evaluated for virus isolation from 626 stool samples of acute flaccid paralysis cases. Poliovirus nonpermissive cells derived from the RD cell line did not show CD155-specific cell-surface immunofluorescence. CD155 gene sequencing confirmed nucleotide base pair deletions within exon2 and exon3. The CD155 knockout RD-SJ40 cells did not support the growth of poliovirus from positive stool samples. All NPEV types were isolated in RD and RD-SJ40 cells. CRISPR correctly edited the CD155 gene of RD cells to render them poliovirus nonpermissive while susceptibility to NPEV remained unchanged. RD-SJ40 cells are safe for NPEV isolation from poliovirus PIM without derogating GAP III containment requirements.

Development of a RT-LAMP assay for detection of SARS-CoV-2.

Background & objectives: The pandemic of SARS-COV-2 began in Wuhan, China in December 2019 and has caused more than 101 million cases worldwide. Diagnostic technologies possessing sensitivity and specificity equivalent to real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assays are needed to ramp up testing capacity in most countries. Newer platforms need to be technically less demanding, require minimum equipment and reduce turn-around time for reporting results. The objective of this study was to exploit loop-mediated isothermal amplification (LAMP) for the detection of SARS-CoV-2 and evaluate its performance by comparison with rRT-PCR. Methods: Reverse-transcription LAMP (RT-LAMP) assay primers were designed to detect envelop (E) and nucleocapsid (N) genes of SARS-CoV-2. Positive control RNA was prepared by in vitro transcription of E and N genes clones. RT-LAMP amplification reactions were incubated at 65°C for 30 min. Results were recorded visually. RT-LAMP results were evaluated by comparing the results obtained with a commercial rRT-PCR kit. Results: The RT-LAMP assay for E and N genes was carried out in separate tubes. RT-LAMP detected about 40 copies of SARS-CoV-2 RNA per reaction. A total of 253 throat swabs were tested using the RT-LAMP assay. The overall diagnostic sensitivity and specificity of the LAMP assay were 98.46 and 100 per cent, respectively, as compared to the rRT-PCR. Interpretation & conclusions: SARS-CoV-2 RT-LAMP assay was designed, standardized and evaluated. The assay showed diagnostic sensitivity and specificity equivalent to rRT-PCR assays. The assay will be useful to increase testing capacity for the detection of SARS-CoV-2 in the country.

Immune Response to SARS-CoV-2 Vaccines.

COVID-19 vaccines have been developed to confer immunity against the SARS-CoV-2 infection. Prior to the pandemic of COVID-19 which started in March 2020, there was a well-established understanding about the structure and pathogenesis of previously known Coronaviruses from the SARS and MERS outbreaks. In addition to this, vaccines for various Coronaviruses were available for veterinary use. This knowledge supported the creation of various vaccine platforms for SARS-CoV-2. Before COVID-19 there are no reports of a vaccine being developed in under a year and no vaccine for preventing coronavirus infection in humans had ever been developed. Approximately nine different technologies are being researched and developed at various levels in order to design an effective COVID-19 vaccine. As the spike protein of SARS-CoV-2 is responsible for generating substantial adaptive immune response, mostly all the vaccine candidates have been targeting the whole spike protein or epitopes of spike protein as a vaccine candidate. In this review, we have compiled the immune response to SARS-CoV-2 infection and followed by the mechanism of action of various vaccine platforms such as mRNA vaccines, Adenoviral vectored vaccine, inactivated virus vaccines and subunit vaccines in the market. In the end we have also summarized the various adjuvants used in the COVID-19 vaccine formulation.

CD155: A Key Receptor Playing Diversified Roles.

Cluster of differentiation (CD155), formerly identified as poliovirus receptor (PVR) and later as immunoglobulin molecule, is involved in cell adhesion, proliferation, invasion and migration. It is a surface protein expressed mostly on normal and transformed malignant cells. The expression of the receptor varies based on the origin of tissue. The expression of the protein is determined by factors involved in the sonic hedgehog pathway, Ras-MEK-ERK pathway and during stressful conditions like DNA damage response. The protein uses an alternate splicing mechanism, producing four isoforms, two being soluble (CD155ß and CD155γ) and two being transmembrane protein (CD155α and CD155δ). Apart from being a viral receptor, researchers have identified CD155 to play important roles in cancer research and the cell signaling field. The receptor is recognized as a biomarker for identifying cancerous tissue. The receptor interacts with molecules involved in the cells' defense mechanism. The immunesurveillance role of CD155 is being deciphered to understand the mechanistic approach it utilizes as an onco-immunologic molecule. CD155 is a non-MHC-I ligand which helps in identifying non-self to NK cells via an inhibitory TIGIT ligand. The TIGIT-CD155 pathway is a novel MHC-I-independent education mechanism for cell tolerance and activation of NK cells. The receptor also has a role in metastasis of cancer and transendothelial mechanism. In this review, the authors discuss the virus-host interaction that occurs via a single transmembrane receptor, the poliovirus infection pathway, which is being exploited as a therapeutic pathway. The oncolytic virotherapy is now a promising modality for curing cancer.

A rapid point of care CC16 kit for screening of occupational silica dust exposed workers for early detection of silicosis/silico-tuberculosis.

Silicosis is an irreversible, incurable and progressive occupational disease caused by prolonged exposure to crystalline-silica dust while working in the relevant industries. Conventionally diagnosis is done by chest radiology, often in an advanced stage as early symptoms often go unnoticed. Early detection and necessary intervention (secondary prevention) could be a realistic possible control strategy for controlling silicosis as no effective treatment is available to stop and/or reverse the pathological process. Additionally, these patients are also vulnerable to pulmonary tuberculosis, which often becomes difficult to treat and with uncertain treatment outcome. Considering India has a huge burden of silicosis and silico-tuberculosis, a rapid and inexpensive screening method was realized to be an urgent need for early detection of silicosis among silica dust exposed workers. Serum club cell protein 16 (CC16) is evidenced to be a useful proxy screening marker for early detection of silicosis as evidenced from the recent research work of ICMR-National Institute of Occupational Health (ICMR-NIOH), India. In this study a lateral-flow assay for semi-quantitative estimation of serum CC16 level was developed. The detection was performed using gold nanoparticles conjugated anti-CC16 monoclonal antibodies. A sum of 106 serum samples was tested to do the performance evaluation of the assay. A concentration of 6 ng/ml or less produced one band, 6.1-9 ng/ml produced two bands, while more than 9 ng/ml produced all the three bands at the test zone. The sensitivity of the assay was 100% while the specificity was 95%. This assay may be used as a sensitive tool for periodic screening of silica dust exposed vulnerable workers for early detection of silicosis in them.

Development of Multiwalled Carbon Nanotube-Based Immunobiosensor for the Detection of Bluetongue Virus.

Bluetongue (BT) disease is a noncontagious disease of domestic and wild ruminants (mainly sheep, cattle, deer) caused by the bluetongue virus (BTV) which is an orbivirus of the Reoviridae family and transmitted by vector Culicoides biting midges. It is a reportable disease of considerable socioeconomic concern and of major importance for the international trade of animals and animal products. Conventional diagnostic methods, such as virus propagation and isolation, immunoassays and also various molecular methods have been developed for the detection of the BTV. Here, we present a novel, rapid and pen-side test for the detection of BTV using multiwalled carbon nanotube (MWCNTs) based immunosensor. Though it is not reported yet. The MWCNTs were prepared, characterized and functionalized with carboxyl group. Viral antibodies were conjugated successfully with functionalized MWCNTs and coated on screen printed carbon electrode (SPCE). These SPCE were evaluated by using electrochemical sensor with an antigen specific to BTV antibodies, resulted in the self-assembled layer of antigen-antibody on the surface of SPCE. The approach described in the present study is a prototype for the development of simple and economic diagnostic tool which will provide the routine screening of BT disease at the door of farmers, thereby increasing the income of farmers by decreasing the cost of diagnosis.

The Importance of Nanomedicine in Prophylactic and Theranostic Intervention of Bacterial Zoonoses and Reverse Zoonoses in the Era of Microbial Resistance.

Emergence of multidrug resistance (MDR), extensively drug resistance (XDR) and pandrug resistance (PDR) strains of bacteria in communicable diseases of zoonotic and reverse zoonotic importance is the major hurdle of one health concept. Increasing level of resistance against antibiotics among bacterial population throughout the world, slow pace of new antibacterial drug discovery and enhanced pace of resistance development by pathogenic bacteria possess major challenges for human and animal health as well as life in future. Alternative management strategy in terms of improved prophylactic vaccine; early, easy and effective diagnostics and therapeutic drugs against those resistant bacteria is the need of the hour. In this context nanomedicine can fit into the multifaceted demands as an effective prophylactic and theranostic alternative to control the communicable diseases in a cost effective manner in the era of microbial resistance. The current review is focused towards delineating the application of nanomaterials as vaccine or drug delivery system, diagnostics and directly acting antimicrobial therapeutic agents in combating the important zoonotic and reverse zoonotic bacterial diseases in recent scenario along with their potential benefits, limitations and future prospects to formulate successful eradication strategies.

Vaccines and Therapies in Development for SARS-CoV-2 Infections.

The current COVID-19 pandemic is caused by the novel coronavirus SARS-CoV-2. The virus causes severe respiratory symptoms which manifest disproportionately in the elderly. Currently, there are over 6.5 million cases and 380,000 deaths reported. Given the current severity of the outbreak, there is a great need for antiviral therapies and vaccines to treat and prevent COVID-19. In this review, we provide an overview of SARS-CoV-2 and discuss the emerging therapies and vaccines that show promise in combating COVID-19. We also highlight potential viral targets that could be exploited by researchers and drug manufacturers.

An Insight into Nanomedicinal Approaches to Combat Viral Zoonoses.

BACKGROUND: Emerging viral zoonotic diseases are one of the major obstacles to secure the "One Health" concept under the current scenario. Current prophylactic, diagnostic and therapeutic approaches often associated with certain limitations and thus proved to be insufficient for customizing rapid and efficient combating strategy against the highly transmissible pathogenic infectious agents leading to the disastrous socio-economic outcome. Moreover, most of the viral zoonoses originate from the wildlife and poor knowledge about the global virome database renders it difficult to predict future outbreaks. Thus, alternative management strategy in terms of improved prophylactic vaccines and their delivery systems; rapid and efficient diagnostics and effective targeted therapeutics are the need of the hour. METHODS: Structured literature search has been performed with specific keywords in bibliographic databases for the accumulation of information regarding current nanomedicine interventions along with standard books for basic virology inputs. RESULTS: Multi-arrayed applications of nanomedicine have proved to be an effective alternative in all the aspects regarding the prevention, diagnosis, and control of zoonotic viral diseases. The current review is focused to outline the applications of nanomaterials as anti-viral vaccines or vaccine/drug delivery systems, diagnostics and directly acting therapeutic agents in combating the important zoonotic viral diseases in the recent scenario along with their potential benefits, challenges and prospects to design successful control strategies. CONCLUSION: This review provides significant introspection towards the multi-arrayed applications of nanomedicine to combat several important zoonotic viral diseases.

Nano-antimicrobials: A New Paradigm for Combating Mycobacterial Resistance.

BACKGROUND: Mycobacterium group contains several pathogenic bacteria including M. tuberculosis where the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) is alarming for human and animal health around the world. The condition has further aggravated due to the speed of discovery of the newer drugs has been outpaced by the rate of resistance developed in microorganisms, thus requiring alternative combat strategies. For this purpose, nano-antimicrobials have emerged as a potential option. OBJECTIVE: The current review is focused on providing a detailed account of nanocarriers like liposome, micelles, dendrimers, solid lipid NPs, niosomes, polymeric nanoparticles, nano-suspensions, nano-emulsion, mesoporous silica and alginate-based drug delivery systems along with the recent updates on developments regarding nanoparticle-based therapeutics, vaccines and diagnostic methods developed or under pipeline with their potential benefits and limitations to combat mycobacterial diseases for their successful eradication from the world in future. RESULTS: Distinct morphology and the underlying mechanism of pathogenesis and resistance development in this group of organisms urge improved and novel methods for the early and efficient diagnosis, treatment and vaccination to eradicate the disease. Recent developments in nanotechnology have the potential to meet both the aspects: nano-materials are proven components of several efficient targeted drug delivery systems and the typical physicochemical properties of several nano-formulations have shown to possess distinct bacteriocidal properties. Along with the therapeutic aspects, nano-vaccines and theranostic applications of nano-formulations have grown in popularity in recent times as an effective alternative means to combat different microbial superbugs. CONCLUSION: Nanomedicine holds a bright prospect to perform a key role in global tuberculosis elimination program.

Development of a rapid test for detection of foot-and-mouth disease virus specific antibodies using gold nanoparticles.

Foot and mouth disease is an economically important transboundary disease of wildlife and cloven hoofed animals including ruminants. In the absence of vaccination, detection of antibodies against structural proteins (SPs) of foot-and-mouth disease virus is an indicator of infection. In the present study, a rapid dot blot assay using gold nanoparticlees was developed for the detection of antibodies against SPs of FMDV. Commercially available FMD vaccine was used as a source of FMD antigen. After the synthesis of gold nanoparticles (GNPs), the GNP-dot blot assay was tested and was found very sensitive, as the detection of antibody was up to 10-4 of serum dilution. The GNP-dot assay was found specific as it didn't give dot with normal horse sera, fetal bovine sera and neonatal bovine calf serum samples when tested at 10-3 working dilution. When 30 serum samples from post-vaccinated buffaloes were tested at dilution of 10-3, all the samples were found positive with the variable intensity of dot. The synthesized GNPs and conjugated GNPS with antibody were characterized for their absorption limit, for their stability and for their approximate size. These characterized conjugated and non-conjugated GNPs were also analyzed by Transmission electron microscopy and Scanning electron microscopy. The GNP dot blot assay developed in this work gave promising results using vaccine antigen and can form an important tool for rapid diagnosis of FMD in FMD free countries, zones free of FMD and during last stage of FMD eradication when FMD vaccination will be withdrawn.

Nanotherapeutics: An insight into healthcare and multi-dimensional applications in medical sector of the modern world.

In recent years nanotechnology has revolutionized the healthcare strategies and envisioned to have a tremendous impact to offer better health facilities. In this context, medical nanotechnology involves design, fabrication, regulation, and application of therapeutic drugs and devices having a size in nano-range (1-100 nm). Owing to the revolutionary implications in drug delivery and gene therapy, nanotherapeutics has gained increasing research interest in the current medical sector of the modern world. The areas which anticipate benefits from nano-based drug delivery systems are cancer, diabetes, infectious diseases, neurodegenerative diseases, blood disorders and orthopedic problems. The development of nanotherapeutics with multi-functionalities has considerable potential to fill the lacunae existing in the present therapeutic domain. Nanomedicines in the field of cancer management have enhanced permeability and retention of drugs thereby effectively targeting the affected tissues. Polymeric conjugates of asparaginase, polymeric micelles of paclitaxel have been recmended for various types of cancer treatment .The advancement of nano therapeutics and diagnostics can provide the improved effectiveness of the drug with less or no toxicity concerns. Similarly, diagnostic imaging is having potential future applications with newer imaging elements at nano level. The newly emerging field of nanorobotics can provide new directions in the field of healthcare. In this article, an attempt has been made to highlight the novel nanotherapeutic potentialities of polymeric nanoparticles, nanoemulsion, solid lipid nanoparticle, nanostructured lipid carriers, dendrimers, nanocapsules and nanosponges based approaches. The useful applications of these nano-medicines in the field of cancer, nutrition, and health have been discussed in details. Regulatory and safety concerns along with the commercial status of nanosystems have also been presented. In summary, a successful translation of emerging nanotherapeutics into commercial products may lead to an expansion of biomedical science. Towards the end of the review, future perspectives of this important field have been introduced briefly.