Mirnalini Sharma

A novel mutation (S227T) in domain II of the envelope gene of Japanese encephalitis virus circulating in North India

Japanese encephalitis (JE) is an important arboviral infection of public health concern. There is a significant variation in mortality (10-30%) in JE viral infection. Epidemics of JE have become regular features in the northern states of India. The recent resurgence of the A226V mutation leading to a widespread Chikungunya epidemic motivated the investigators to search for any such mutational occurrence with Japanese encephalitis virus (JEV) isolated from this region. This study looked for mutation of clinical strains at amino-acid positions 176, 177, 227, 244, 264 and 279. A novel mutation S227T was detected corresponding to the loop region of domain II, E gene of JEV in comparison to Indian and other isolates from different parts of the world. Genotype III was found to be circulating in this geographical area. Further studies are required to ascertain its role in JE pathogenesis and vector competency.

Utility of multiplex reverse transcriptase-polymerase chain reaction for diagnosis and serotypic characterization of dengue and chikungunya viruses in clinical samples ☆

The reemergence of chikungunya virus (CHIKV) has compounded the already existing dengue problem because of clinical similarities and common vector, demanding the need for a rapid and specific diagnosis. Thus, dengue chikungunya multiplex reverse transcriptase-polymerase chain reaction (DCmRT-PCR) was developed and validated for simultaneous detection of dengue and chikungunya viral infections and its utility in virus serotyping. Blood samples from 97 suspected dengue and chikungunya cases and 10 healthy controls were subjected to dengue and chikungunya conventional RT-PCR and DCmRT-PCR. Thirty-one of 97 samples were positive for dengue or chikungunya viral RNA by RT-PCR and DCmRT-PCR with 100% concordance. DCmRT-PCR products were cycle sequenced. Seven dengue virus strains were clustered within genotype III of DENV-3 and 4 within genotype III of DENV-1, whereas chikungunya sequences were clustered within the Central/East African genotype. DCmRT-PCR was found to be a potential rapid test for simultaneous detection of dengue and CHIKV in clinical samples along with dengue serotyping.

Clinical applicability of single-tube multiplex reverse-transcriptase PCR in dengue virus diagnosis and serotyping

This study has evaluated the clinical applicability of a single‐tube multiplex RT‐ PCR as compared with a two‐step nested RT‐PCR for the diagnosis as well as serotyping of dengue virus in patients samples. Seventy‐six acute phase blood samples collected from clinically suspected dengue patients during the 2008 outbreak were subjected to two‐step nested RT‐PCR and single‐tube multiplex RT‐PCR for dengue diagnosis and serotyping. Of the 76 samples, 17 (22.4%) were positive for dengue viral RNA. Single dengue virus infection was found in 16 cases and 1 had concurrent infection with two serotypes (3&1). Dengue serotype 3 was the predominant serotype (70.5%), followed by serotype 1 (23.5%). Single‐tube multiplex PCR had concordant result with that of two‐step nested RT‐PCR including the one with concomitant infection. This study reveals the predominance of dengue serotype 3 in North India in addition to the co‐circulation of multiple serotypes and concomitant infection. The rapid and accurate diagnostic capability of single‐tube multiplex RT‐PCR used in the study appears to be promising enough to be commonly used for dengue viral detection as well as serotyping. J. Clin. Lab. Anal. 25:76–78, 2011.

Ribonucleic acid extraction from archival formalin fixed paraffin embedded myocardial tissues for gene expression and pathogen detection.

Archival tissue samples preserved in formalin are a great source of treasure for biomedical research and diagnostics. Formalin, though is a good preservative, causes the modification of nucleic acid limiting the application of fixed tissues. The present study evaluated three methods of RNAextraction for constitutive gene expression and pathogen detection.

Clinical applicability of various dengue diagnostic tests in resource-limited endemic settings

Introduction: Dengue is one of the most important arboviral infections caused by one of the four dengue serotypes, 1-4. Objective: To study the applicability of different diagnostic methods in diagnosis of dengue viral infection. Materials and Methods: A total of 2101 blood samples were collected for confirmation of dengue viral infection. All the samples were tested by dengue-specific IgM ELISA, of which 111 were also tested for NS1 antigen detection and 27 acute samples (≤5 days) were further subjected for viral RNA detection by RT-PCR and isolation in C6/36 cell line. To detect the sensitivity of NS1 antigen for different dengue virus serotypes, four dengue serotype 1 and 12 dengue 3 were subjected for the NS1 antigen assay. Results: Most common age group affected was 16-45 years, with male to female ratio of 2.8:1. During first 3 days of illness virus isolation and RT-PCR were the most sensitive (83%) followed by NS1 antigen detection (75%) and IgM detection (37.5%). The positivity of IgM detection was found to be significantly higher as compared to NS1 detection during 4 to 5 days and also after 5 days of illness (P < 0.05). Dengue serotypes 1 and 3 were found to be co-circulated, dengue 1 being the predominant serotype. Conclusion: Virus isolation and RT-PCR were the most sensitive tests during the early period of illness whereas beyond third day, IgM antibody detection was found to be the most sensitive method of dengue diagnosis.

Disseminated coxsackievirus B fulminant myocarditis in an immunocompetent adult: A case report: Case of disseminated coxsackievirus myocarditis

Coxsackieviral myocarditis is associated with systemic involvement in neonates; however, fulminant coxsackieviral myocarditis is rare in adults, and its dissemination with fatal myocarditis involving kidneys, liver, and adrenal is further rarely reported. We report a case of fulminant myocarditis along with dissemination of coxsackievirus, which was clinically unrecognized.

Inhibition of coxsackievirus infection in cardiomyocytes by small dsRNA targeting its cognate coxsackievirus adenovirus receptor

Background & objectives: Coxsackievirus B (CVB), a member of human Enterovirus group, is the most common cause of viral myocarditis. Coxsackievirus adenovirus receptor (CAR) is identified as a key determinant for the entry of CVB in the target cells. Thus, blockade of receptor by RNA interference (RNAi) may inhibit the entry and pathogenesis of CVB in cardiac cells. The present study was aimed to determine the effect of CAR small dsRNA (siRNA) on coxsackieviral load and CAR expression in coxsackievirus-infected cardiomyocytes. Methods: Transfection efficiency in rat cardiomyocytes (H9c2) was determined by the fluorescent microscopy and flow cytometry. CAR siRNA dose was optimized based on cell viability and relative CAR messenger RNA (mRNA) expression. Cardiomyocytes were transfected with CAR siRNA followed by infection with 100 multiplicity of infection of CVB, which were harvested after 24, 48 and 72 h post-infection (p.i.). RNA was extracted for relative CAR mRNA expression. Cells were freeze-thawed thrice for estimating coxsackieviral load. Results: The efficiency of transfection was optimized to be >80 per cent and CAR siRNA dose of 60 pmol was standardized. The knockdown of CAR by siRNA decreased its expression twice the expression in normal cardiomyocytes after 24 h p.i. of CVB. The treatment with CAR siRNA resulted in significant two log reduction of CVB load in cardiomyocytes infected with CVB at 24 h p.i. and retained till 72 h p.i. Interpretation & conclusions: The inhibition of CAR by siRNA was found to be effective against CVB in cardiomyocytes. However, this treatment strategy has to be evaluated in vivo to develop a new treatment strategy for patients suffering with viral myocarditis.