Isolation and characterization of emerging Mpox virus from India.

Mpox (previously known as Monkeypox) has recently re-emerged, primarily through human-to-human transmission in non-endemic countries including India. Virus isolation is still considered as the gold standard for diagnosis of viral infections. Here, the qPCR positive skin lesion sample from a patient was inoculated in Vero E6 cell monolayer. Characteristic cytopathic effect exhibiting typical cell rounding and detachment was observed at passage-02. The virus isolation was confirmed by qPCR. The replication kinetics of the isolate was determined that revealed maximum viral titre of log 6.3 PFU/mL at 72 h postinfection. Further, whole genome analysis through next generation sequencing revealed that the Mpox virus (MPXV) isolate is characterized by several unique SNPs and INDELs. Phylogenetically, it belonged to A.2 lineage of clade IIb, forming a close group with all other Indian MPXV along with few from USA, UK, Portugal, Thailand and Nigeria. This study reports the first successful isolation and phenotypic and genotypic characterization of MPXV from India.

Simple and field amenable loop-mediated isothermal amplification-lateral flow dipstick assay for detection of west Nile virus in human clinical samples.

AIM: West Nile encephalitis caused by infection with the West Nile virus (WNV) is endemic in many regions of the world and is a global public health threat. The aim of this report was to develop a method using colorimetry-based reverse-transcription loop-mediated isothermal amplification (cRT-LAMP) and RT-LAMP combined with lateral-flow dipstick (LFD) for rapidly detecting WNV in low-infrastructure settings. METHODS AND RESULTS: The primers for the cRT-LAMP and RT-LAMP-LFD assays were designed based on env gene of the WNV. Primers concentration, temperature and time were optimized for cRT-LAMP and RT-LAMP-LFD. The diagnostic performance of the cRT-LAMP and RT-LAMP-LFD assays was evaluated using human serum samples from 110 patients who were clinically suspected to be infected with WNV. The RT-LAMP was performed in a heating block at 63°C for 40 min. The LAMP amplicons were visible in the lateral-flow dipstick within 5 min. The detection limit of the developed cRT-LAMP and RT-LAMP-LFD assays was 10 copies and this assay showed a high degree of specificity for WNV. Compared with quantitative real-time RT-PCR assay, the kappa value of cRT-LAMP and RT-LAMP-LFD were 0.970. CONCLUSIONS: These results showed that the newly developed WNV-specific cRT-LAMP and RT-LAMP-LFD assays can be employed as an alternative method for screening of WN-suspected human samples. The results revealed that the assay could potentially identify the virus without interference from human serum samples. Collectively, all results revealed that cRT-LAMP and RT-LAMP-LFD assays offer a suitable field-based diagnosis of WNV. SIGNIFICANCE AND IMPACT OF THE STUDY: The cRT-LAMP and LAMP-LFD platform for the detection of WNV is rapid, accurate and simple-to-perform. Our present method has not only a short turnaround time but also avoided cross-contamination problem. Moreover, the use of simple lateral flow dipsticks broadens its application potential for the point-of-care use in resource-limited settings during outbreak situations. To the best of our knowledge, this is the first report for the development of cRT-LAMP and LAMP-LFD assays for rapid, simple, specific and sensitive detection of WNV using human clinical samples and EvaGreen dye.

One-step single-tube accelerated quantitative nucleoprotein gene-specific reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assay for rapid, real-time & reliable clinical detection of Ebola virus.

Background & objectives: Due to the absence of specific drugs or vaccines for Ebola virus disease, rapid, sensitive and reliable diagnostic methods are required to control the transmission chain of the disease and for better patient management. Isothermal amplification of nucleic acids has emerged as a promising alternative in which rapid and efficient amplification is achieved at a constant temperature without the thermal cycling required in PCR. Methods: A one-step single-tube accelerated quantitative reverse trascription loop-mediated isothermal amplification (RT-LAMP) assay was developed by targeting the NP gene of 2014 Zaire Ebola virus (ZEBOV). The RT-LAMP assay was found to be specific for ZEBOV, without having any cross-reactivity with related haemorrhagic fever viral agents. Results: The comparative evaluation of Ebola virus NP gene-specific RT-LAMP assay with reverse transcription (RT) - PCR and TaqMan real-time RT-PCR demonstrated that RT-LAMP was 10-1000 folds more sensitive than TaqMan real-time RT-PCR and conventional RT-PCR, respectively, with a detection limit of 1 copy number. In the absence of real-world clinical samples, the feasibility of Ebola virus RT-LAMP assay for clinical diagnosis was evaluated with different body fluids including serum, urine, saliva, semen and stool samples from healthy human volunteers spiked with gamma-irradiated ZEBOV 2014 obtained from Robert Koch Institute, Berlin, Germany, through the European Network for Diagnostics of Imported Viral Diseases. The Ebola virus RT-LAMP assay could correctly be picked up the spiked samples up to 1 copy of viral RNA without having any matrix interference. The monitoring of gene amplification can also be visualized with the naked eye by using SYBR Green I fluorescent dye. Interpretation & conclusions: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool as a point-of-care diagnosis for the rapid and real-time detection of Ebola virus in resource-limited healthcare settings of developing countries.

Surface plasmon resonance sensing of Ebola virus: a biological threat.

Here, different monoclonal antibodies (mAb1, mAb2 and mAb3) of Ebola virus were screened in a real-time and label-free manner using surface plasmon resonance (SPR) to select an appropriate antibody for biosensor applications against a biological warfare agent. For this purpose, a gold SPR chip was modified with 4-mercaptobenzoic acid (4-MBA), and modification was confirmed by FTIR-ATR and EIS. The 4-MBA-modified gold SPR chip was used for immobilization of the recombinant nucleoprotein of Ebola (EBOV-rNP), and the interactions of mAb1, mAb2 and mAb3 were then investigated to determine the best mAb based on the affinity constant (KD), expressed as equilibrium dissociation constant. KD values of 809 nM, 350 pM and 52 pM were found for the interaction of mAb1, mAb2 and mAb3 of Ebola with the immobilized EBOV-rNP, respectively, thus reflecting the high affinity of mAb3. This was confirmed by ELISA results. The thermodynamic parameters (ΔG, ΔH and ΔS) for the interaction between mAb3 and EBOV-rNP were also determined, which revealed that the interaction was spontaneous, endothermic and driven by entropy. The SPR limit of detection of EBOV-rNP with mAb3 was 0.5 pg ml-1, showing mAb3 to be the best high-affinity antibody in our study. This study has opened up new possibilities for SPR screening of different monoclonal antibodies of BWA through the convergence of materials science and optical techniques.

Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus.

Background & objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV. Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection. Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively. Interpretation & conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients.

Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV. Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection. Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.

Emergence of influenza A (H1N1)pdm09 genogroup 6B and drug resistant virus, India, January to May 2015.

To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients.

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples.

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.

BACKGROUND & OBJECTIVES: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. METHODS: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. RESULTS: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. INTERPRETATION & CONCLUSION: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Development and evaluation of indirect antibody ELISA assay for early diagnosis and surveillance of Crimean-Congo hemorrhagic fever infection in humans.

Crimean-Congo hemorrhagic fever (CCHF) is an important tick borne zoonotic viral disease of humans. CCHF virus causes sporadic cases of severe illness across a huge geographic area across Africa to Europe to Asia including India. CCHF has emerged as a major public health concern in western Indian states including Gujarat and Rajasthan, where regular human cases were reported since the year 2011. Human serve as the dead-end host, and they gain infection via infected tick bite, in close contact with ruminants and from slaughter house. Currently, the detection of this fatal infection is limited to BSL-4 laboratory which is scarce even in developed economies. Thus, a safe, sensitive assay for early immunodiagnosis is crucial for disease management and containing the outbreak. In this study, the conserved recombinant nucleoprotein was exploited as a safe, scalable alternate antigen for development of indirect IgM and IgG ELISA detection platform. The indirect ELISA was evaluated using suspected clinical samples collected from hotspot areas in India. Comparison with reference MAC ELISA and IgG ELISA revealed a correlation of 95% and 100% respectively. The results indicate that the developed IgM and IgG indirect ELISA has high sensitivity and specificity for detecting CCHFV antibodies among human. These assays are easy to perform and can be employed for high throughput screening of human samples for clinical diagnosis as well as serosurveillance. These assays are also amenable for conversion to low-cost point of care testing formats for application in resource limited settings.

Development of a Reverse Transcription Loop - Mediated Isothermal Amplification [RT-LAMP] as a early rapid detection assay for Crimean Congo Hemorrhagic Fever virus.

Presently diagnosis of Crimean Congo Hemorrhagic Fever virus (CCHFV) infection relies on real-time and end-point RT-PCR, and serodiagnostic assay. These assays are time consuming and cannot be used as a routine screening test. The objective of this study was to develop a rapid diagnostic test that could be completed in < 60 minutes. Rapid detection of CCHFV infection is important for faster delivery of appropriate therapeutics, clinical management of patient and also important to contain the outbreak. In the present study, we have developed a rapid and sensitive single tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of CCHFV. The limit of detection of RT-LAMP vis-a-vis Real-time RT-PCR assay is 10 RNA copies. Further, CCHFV specific RT-LAMP assay was successfully evaluated with human and tick samples. The assay correctly picked up diverse CCHFV isolates indicating its applicability for different strains. A comparative evaluation of the RT-LAMP assay vis-à-vis with the real-time RT-PCR revealed 100% concordance with 100 % sensitivity and specificity respectively. No cross reactivity with related Flaviviruses and hemorrhagic fever viruses was observed. The assay is a rapid, isothermal, simple to perform molecular diagnostic, which can be performed in a portable heating block device. CCHF RT-LAMP assay can be used in low resource laboratories for monitoring of CCHFV outbreaks in remote rural regions in affected countries.

Development and application of a recombinant Envelope Domain III protein based indirect human IgM ELISA for Kyasanur forest disease virus.

Kyasanur forest virus disease (KFD) is a major public health concern in India. Its etiology KFD virus causes haemorrhagic fever with severe sequelae in humans. Due to continuous spatiotemporal expansion of KFD in last decade, the incidences of positive cases have been increasing in both humans and primates. Early diagnosis is of prime importance for disease management and epidemiological containment. In the present study, the highly immunogenic Envelope Domain III (EDIII) antigen was produced using prokaryotic expression system with an yield of 8 mg/L. The protein was purified using affinity chromatography and confirmed for its immuno-reactivity by western blot and UPLCMS/MS analysis. The recombinant EDIII was used as an antigen for the standardization of ELISA to detect anti KFD IgM antibodies in humans. The ROC curve was prepared to set the optimum cut-off OD for the assay. The comparative evaluation of the assay with a reference MAC ELISA revealed 86.96% concordance, 82.22% sensitivity and 91.48% specificity. Inter-rater agreement was performed with kappa index revealing significant agreement between the assays. This is the first study using safe recombinant protein antigen-based detection of anti KFDV antibodies in humans. This simple and scalable ELISA assay will be applicable for large scale screening of samples for combating the emerging threats of KFD in newer territories.

Monoclonal antibody-based antigen capture immunoassay for detection of circulating non-structural protein NS1: implications for early diagnosis of Japanese encephalitis virus infection.

An early diagnosis of Japanese encephalitis (JE) is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. The NS1 antigen has an advantage over IgM enzyme-linked immunosorbent assay (ELISA) for early confirmatory diagnosis of Japanese encephalitis virus (JEV) infection due to its proliferation on the surface of the host cells in the acute phase of infection. In this study, the development and evaluation of JE-specific NS1 antigen capture ELISA is described using high-affinity monoclonal antibody specific to the recombinant NS1 protein for early diagnosis of JE. The gene encoding NS1 protein was cloned and expressed in the pQE30UA expression vector followed by purification of the recombinant protein by affinity chromatography. A sandwich ELISA for antigen detection was developed using purified rabbit IgG antibody and mouse monoclonal antibody as the capture and detector antibody, respectively. The application of JE NS1 antigen ELISA for early diagnosis was evaluated with 120 acute phase sera and 80 CSF samples. The comparative evaluation of the JE NS1 antigen ELISA by real-time RT-PCR revealed 97% concordance with a sensitivity and specificity of 97% and 98%, respectively. The JE NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. These findings suggest that the JEV NS1 antigen capture ELISA may help early diagnosis of JE infection.

Development and evaluation of reverse transcription loop-mediated isothermal amplification for rapid and real-time detection of Kyasanur forest disease virus.

SIGNIFICANCE: Kyasanur forest disease (KFD), a re-emerging tick-borne viral disease, causes severe hemorrhagic fever in humans and nonhuman primates. KFD virus (KFDV) is a member of the genus Flavivirus. KFD is now increasingly reported outside its endemic zone in India. Rapid and specific detection of the KFDV plays a critical role in containment of the outbreak. The diagnosis of KFD currently relies on real-time RT-PCR, nested RT-PCR, end point RT-PCR, and serodiagnostic assay. These assays are tedious, time-consuming, and cannot be used as a routine screening platform. OBJECTIVE: The present study was aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for molecular diagnosis of KFD. DESIGN: The gene amplification reaction was accomplished by incubation at a constant temperature of 63°C for 60min. RESULTS: The limit of detection of RT-LAMP assay was 10 copies. KFD RT-LAMP assay was successfully evaluated with diverse host samples including humans, monkeys, and tick. The assay correctly picked up different KFD isolates indicating its applicability for divergent strains. Comparative evaluation of RT-LAMP assay with quantitative TaqMan real-time RT-PCR revealed 100% concordance. No cross-reaction with related flavi and other hemorrhagic fever viruses was observed, indicating its high specificity. CONCLUSION AND RELEVANCE: The RT-LAMP test developed in this study will serve as a rapid, sensitive alternate detection method for KFDV infection and would be useful for high throughput screening of clinical samples in resource limited areas during outbreaks.

Development of double antibody sandwich ELISA as potential diagnostic tool for rapid detection of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) virus, a highly pathogenic viral agent is responsible for severe fatal hemorrhagic infections in many parts of the world. The early diagnosis of CCHF infection is important for successful clinical management and epidemiological control. The nucleoprotein (NP) of CCHFV being highly conserved and immunogenic is used as early diagnostic marker. In this study, we report a rapid and sensitive double antibody based antigen capture ELISA to detect Crimean-Congo hemorrhagic fever virus (CCHFV). Highly specific polyclonal and monoclonal antibody against NP has been generated and used as capture and detector antibody respectively. The assay was able to detect viral nucleoprotein in different matrices including human serum, ticks and culture supernatant. The detection limit of the developed sandwich ELISA assay was 25 ng of purified antigen. Comparison with a real time RT-PCR revealed its detection limit to be 1000 genome equivalents of CCHFV. Further the assay was comparatively evaluated with a commercial kit employing gamma irradiated CCHFV, revealing a sensitivity and specificity of 100%. This newly developed sandwich ELISA (sELISA) with high sensitivity and specificity could be used as an efficient method for the detection of CCHF virus in humans, ticks and culture supernatant. The assay will be useful as alternate tool for diagnosis of acute infection and is amenable for screening of large scale samples in resource limited settings.

Emergence and expansion of highly infectious spike protein D614G mutant SARS-CoV-2 in central India.

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock.

Polymerase Spiral Reaction Assay for Rapid and Real Time Detection of West Nile Virus From Clinical Samples.

West Nile virus (WNV) is a mosquito-borne virus of public health importance. Currently, there is no FDA approved vaccine available against WNV infection in humans. Therefore, the early diagnosis of the WNV infection is important for epidemiologic control and timely clinical management in areas where multiple Flaviviruses are endemic. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that rapidly and accurately detects the envelope (env) gene of WNV. RT-PSR assay was optimized at 63°C for 60 min using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The standard curve for RT-PSR assay was generated using the 10-fold serial dilutions of in vitro transcribed WNV RNA. To determine the detection limit of RT-PSR assay, an amplified product of conventional RT-PCR was in vitro transcribed as per standard protocol. The detection limit of the newly developed RT-PSR assay was compared with that of conventional RT-PCR and CDC reported TaqMan real-time RT-PCR using a serial 10-fold dilution of IVT WNV RNA. The detection limit of RT-PSR was found to be 1 RNA copy, which is 100-fold higher than that of conventional RT-PCR (100 copies). This suggests that RT-PSR assay is a valuable diagnostic tool for rapid and real-time detection of WNV in acute-phase serum samples. The assay was validated with a panel of 107 WNV suspected human clinical samples with signs of acute posterior uveitis and onset of febrile illness. Out of 107 samples, 30 were found positive by RT-PSR assay. The specificities of the selected primer sets were established by the absence of cross-reactivity with other closely related members viruses of the Flaviviruses, Alphaviruses, and Morbilliviruses groups. No cross-reactivity was observed with other viruses. To best of our knowledge, this is the first report describing the RT-PSR assay for the detection of RNA virus (WNV) in clinical samples. RT-PSR is a high throughput method and more than 30 reactions can be run at once in real-time turbidimeter. PSR assay has potential to be used for a rapid screening of large number of clinical samples in endemic areas during an outbreak.

Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples.

West Nile virus (WNV) causes West Nile fever and encephalitis worldwide. Currently, there are no effective drugs or vaccines available in the market to treat WNV infection in humans. Hence, it is of paramount importance to detect WNV early for the success of the disease control programs and timely clinical management in endemic areas. In the present paper, we report the development of real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for rapid and real-time detection of WNV targeting the envelope (env) gene of the virus. The RPA reaction was performed successfully at 39°C for 15 min in a real-time thermal cycler. The sensitivity of this assay was found similar to that of the quantitative real-time RT PCR (RT-qPCR) assay, which could detect 10 copies of the gene. The efficacy of the assay was evaluated with a panel of 110 WN suspected human samples showing the signs of retinitis, febrile illness and acute posterior uveitis. In comparison with RT-qPCR, RT-RPA showed a specificity of 100% (CI, 95.07-100%) and sensitivity of 96.15% (CI, 80.36-99.90%) with a negative (NPV) and positive predictive value (PPV) of 98.65 and 100%, respectively. The level of agreement between RT-RPA and reference RT-qPCR assay was shown to be very high. The turnaround time of real-time RPA assay is about 10-20 times faster than the RT-qPCR, which confirms its utility in the rapid and sensitive diagnosis of WNV infection. To the best of our knowledge, this is the first report which deals with the development of real-time RT-RPA assay for simple, rapid, sensitive, and specific detection of WNV in human clinical samples. The present RT-RPA assay proves to be a powerful tool that can be used for the rapid diagnosis of a large number of patient samples in endemic settings.

Vector derived artificial miRNA mediated inhibition of West Nile virus replication and protein expression.

West Nile virus (WNV) has been found to be a common cause of neuroinvasive arboviral disease worldwide in human and horses. The process of RNA interference induced by small RNA molecules, like small interfering RNA (siRNA) and microRNA (miRNA), proved to be a novel approach for preventing viral infections. So far there is no published data for inhibition of West Nile virus by vector delivered artificial miRNA which believed to have more inhibitory potential than small interfering (siRNA). In the present study, we designed two artificial miRNA (amiRNAs) targeting the conserved NS5 and NS2A genomic regions of West Nile virus. These amiRNAs oligos were cloned in to miRNA based vector having murine miR-155 backbone which allows the high expression of amiRNAs in green fluorescent protein (GFP) tagged form. Vero cells were transiently transfected by cytomegalovirus (CMV) promoter derived vector expressing amiRNAs transcribed by RNA Pol II. Efficacy of amiRNA targeting the NS5 and NS2A regions of WNV was determined in highly virulent WNV Eg101 strain in Vero cells. The result indicated that both amiRNA effectively inhibit West Nile virus replication. The concatenated amiRNA having dual pre-amiRNA expression cassette showed better efficacy. amiRNA targeting NS5 showed best protection against WNV infection and percentage reduction of WNV titer was observed at 96 hpi is 97.11%. Further study for inhibition of WNV replication was assessed by plaque assay, quantitative reverse transcriptase PCR (qRT-PCR) assay, Immunofluorescence assay and Western blot analysis. Present study concludes that amiRNA (NS5) targeting conserved region of gene significantly reduced the virus replication as determined by plaque assay. Similarly, reduction was also observed at RNA and protein level through real-time RT-PCR and Western blot analysis directly correlate with the inhibition of WNV replication. Here, we describe our current understanding of the role of miRNAs in host defense response against West Nile virus, as well as their potential as new therapeutic approaches.