Nagesh K. Tripathi

Rapid and Real-Time Detection of Chikungunya Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay

ABSTRACT The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 × 108 to 2 × 102 copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 × 108 to 2 × 101 copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63°C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.

Japanese Encephalitis Outbreak, India, 2005

An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid and Real-Time Detection of Japanese Encephalitis Virus

ABSTRACT The standardization and validation of a one-step, single-tube accelerated quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is reported for rapid and real-time detection of Japanese encephalitis virus (JEV). The RT-LAMP assay reported in this study is very simple and rapid; the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of six primers targeting the E gene of JEV. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared to that of RT-PCR, with a detection limit of 0.1 PFU. The specificities of the selected primer sets were established by cross-reactivity studies with other closely related members of the JEV serocomplex as well as by evaluation of healthy human volunteers. The comparative evaluation of the RT-LAMP assay for clinical diagnosis with a limited number of patient cerebrospinal fluid samples revealed 85% concordance with conventional RT-PCR, with a sensitivity and a specificity of 100% and 86%, respectively. The concentration of virus in most of the clinical samples was 102 to 105 PFU/ml, as determined from the standard curve based on the time of positivity in the samples. In addition, the monitoring of gene amplification can also be visualized with the naked eye by using SYBR green I fluorescent dye. Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool for the rapid and real-time detection of JEV not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.

Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection

PURPOSE Dengue is one of the most serious mosquito-borne viral infections affecting tropical and subtropical countries in the world. Since there is no immunoprophylactic or specific antiviral therapy available, timely and rapid diagnosis plays a vital role in patient management and implementation of control measures. This paper evaluates a commercially available NS1 antigen capture ELISA vis-a-vis SD bioline Dengue NS1 antigen test for early detection of dengue virus. MATERIALS AND METHODS To evaluate a commercial NS1 antigen detection kit vis-a-vis SD bioline Dengue NS1 antigen test, a total of 91 clinical samples were tested. Virological investigations with regard to dengue virus, viz. NS1 antigen capture ELISA (Panbio, Australia), SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were performed. RESULTS Out of 91 samples, 24 (26%) were positive by NS1 antigen capture ELISA, 15 (16%) by SD bioline Dengue NS1 antigen test and 11(12%) positive by RT-PCR analysis. The RT-PCR-positive samples were further subjected to virus isolation and resulted in three isolates. The results of the Panbio NS1 antigen capture ELISA, SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were correlated among themselves. CONCLUSIONS The present study comprehensively established the utility of NS1 antigen ELISA in early diagnosis of dengue infection.

Production and characterization of recombinant dengue virus type 4 envelope domain III protein.

Dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. Cultivation process for production of recombinant dengue virus type 4 envelope domain III (rDen 4 EDIII) protein in Escherichia coli was developed for its diagnostic use as well as for further studies in immunoprophylaxis. The dissolved oxygen level was maintained at 20-30% of air saturation. The culture was induced with 1mM of isopropyl beta-d-thiogalactoside when dry cell weight was 13.78 g l(-1) and cells were further grown for 4h to reach 17.31 g l(-1) of culture. The protein was overexpressed in the form of insoluble inclusion bodies. The rDen 4 EDIII protein was purified by affinity chromatography and analyzed by SDS-PAGE. The final yield of purified rDen 4 EDIII protein in this method was approximately 196 mg l(-1) of culture. The purified protein was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue infected human serum samples. These results show that the product has the potential to be used for the diagnosis of dengue infection or for further studies in vaccine development. This production system may also be suitable for the high yield of other recombinant dengue proteins.

Production, purification and characterization of recombinant dengue multiepitope protein

Dengue is an acute mosquito‐borne viral disease of humankind. Dengue fever, dengue haemorrhagic fever and dengue shock syndrome have become global public health problems in recent years. rDME‐G (recombinant dengue multiepitope protein that can specifically detect IgG) was produced in a 5‐litre fermenter in Escherichia coli for use in diagnosis. The culture was induced with 1 mM isopropyl β‐d‐thiogalactoside and cells were further grown for 4 h before harvesting. After fermentation, dry cell weight resulted in approx. 16.2 g/l. The rDME‐G protein was purified from inclusion bodies using affinity chromatography. The final yield of purified rDME‐G protein from fermentation resulted in approx. 168 mg/l of pure biologically active rDME‐G protein. The purity of rDME‐G protein was checked by SDS/PAGE analysis and the reactivity of this protein was further determined by Western blotting. The purified protein was used to develop an in‐house dipstick ELISA and tested using a panel of 60 patient sera characterized using the commercially available tests for detection of dengue antibody. We compared our results with IgG‐capture ELISA (Pan‐Bio, Windsor, QLD, Australia) and rapid IC (immuno‐chromatography) test (Pan‐Bio). By using rDME‐G protein as an antigen, in the dipstick ELISA, the results were in excellent agreement with commercial rapid IC test and IgG capture ELISA. These results show that the product has a promising potential to be used for diagnosis of dengue in both laboratory‐ and field‐based detection systems with minimum cost and a high degree of sensitivity and specificity.

Cloning and expression of domain III of the envelope gene of Japanese encephalitis virus: evaluation for early clinical diagnosis by IgM ELISA.

Japanese encephalitis virus (JEV) is the single largest cause of childhood viral encephalitis in the world with an estimated 50,000 cases and 10,000 deaths annually. The laboratory diagnosis is based essentially on IgM ELISA owing to low transient viremia making virus isolation difficult. In addition the requirement of cerebrospinal fluid (CSF) sample for confirmatory molecular diagnosis by reverse transcription-polymerase chain reaction (RT-PCR) makes IgM ELISA the test of choice for early clinical diagnosis. The development and evaluation of a highly sensitive and specific IgM ELISA using the recombinant domain III envelope protein (rJEV-DIII) for rapid, early and accurate diagnosis of JEV is reported in the present study. The gene coding for the envelope protein of JEV was cloned and expressed in pET 30a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rJEV-DIII was optimized that had no reactivity with healthy persons. The comparative evaluation accomplished with the JE-Dengue IgM Combo ELISA (PanBio, Brisbane, Australia) and JEV Chex (XCyton Diagnostic Ltd., Bangalore, India) ELISA kits, by subjecting 120 acute phase of clinical samples revealed more than 95% accordance. The rJEV-DIII ELISA and the PanBio ELISA were found to have a sensitivity and specificity of 98% and 96%, respectively. The compared positivity of the rJEV-DIII ELISA and SYBR green-I based real-time RT-PCR assay in CSF samples revealed higher positivity. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. The recombinant domain III envelope protein based JEV specific ELISA will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli.

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed‐batch cultivation process were also developed in optimized medium. After fed‐batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ∼649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS–PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.

Production of IgM Specific Recombinant Dengue Multiepitope Protein for Early Diagnosis of Dengue Infection

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME‐M) protein specific to IgM in E. coli was produced in a 5‐L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME‐M protein was purified under denaturing conditions using single‐step nickel nitrilotriacetate (Ni‐NTA) affinity chromatography. The final yield of purified rDME‐M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME‐M protein was checked by SDS‐PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme‐linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in‐house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan‐Bio) and rapid immunochromatography (IC) test (Pan‐Bio). These results show that the in‐house dipstick ELISA using rDME‐M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein.

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freunds complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.