A shift in circulating rotaviral genotypes among hospitalized neonates.

In neonates, rotavirus (RV) infection is generally nosocomial. The control of rotaviral infection within hospital settings is challenging due to prolonged shedding of the virus and contamination of the surrounding environment. There are few studies that have reported asymptomatic infection within neonates. In this study, neonates were screened for RV infection and possible clinical manifestations that may play a role in RV acquisition were analysed. Stool samples were collected from 523 hospitalized neonates admitted for > 48 h in a low-cost and higher-cost tertiary centre. RV antigen was screened using ELISA and the samples which tested positive were confirmed by semi-nested RT-PCR. RV was detected in 34% of participants and genotypes identified included G12P[11] (44.4%), G10 P[11] (42.6%), G10G12P[11] (10.1%) and G3P[8] (2.9%). ICU admissions were associated with higher viral shedding (p < 0.05). Hospitalization in the low-cost facility ICU was associated with higher RV acquisition risk (p < 0.05). RV was detected in higher rates (36.9%) among neonates with gastrointestinal manifestations. G10P[11] was the predominant genotype for several years (1988-2016) among neonates within India. The preponderance of an emerging G12P[11] genotype and heterotypic distribution was documented. RV surveillance is important to identify emerging strains and establish the road ahead in managing RV infection.

Detection of cytomegalovirus disease by real-time quantitative PCR targeting immediate early gene (ppUL83) in different samples among post-renal-transplant recipients.

Renal transplantation is a treatment option for end-stage renal disease (ESRD). Cytomegalovirus (CMV) infection was analysed among symptomatic and asymptomatic post-renal-transplant recipients (PRTRs). A total of 30 PRTRs were enrolled. DNA was extracted and quantitative real-time PCR for CMV (CMV R-Gene, France) targeting ppUL83 gene was performed on whole blood, urine and saliva. The detection rate of CMV was found to be 27% (n = 8) in different samples, including whole blood, urine and saliva. Among 30 PRTRs, 53% (n = 16) of the PRTRs did not shed virus in saliva. About 7% of CMV was detected only in saliva among PRTRs who were symptomatic.

Genetic characterization of Chikungunya virus 2009 isolates from South India.

Chikungunya Virus (CHIKV) is a single stranded positive sense enveloped RNA virus. Re-emergence of CHIKV caused a massive outbreak with severe clinical manifestation affecting multiple organs. The genetic diversity of CHIKV, which caused recurring outbreaks in India, was studied. Blood samples were collected from suspected human cases of CHIKV infection in Chennai, Tamil Nadu and three Northern districts of Kerala in Southern India during the CHIKV outbreak in 2009. A partial E2 gene segment was amplified by RT-PCR. Among 119 samples 37 samples were positive for CHIKV by RT-PCR. Phylogenetic analysis revealed that the isolated sequences belonged to Indian Ocean Lineage (IOL) of ECSA genotype. The mutational analysis revealed the presence of substitutions such as S299N, T312M, A344T, S375T, V386G, W339R and S375P in the current study. In addition, a novel mutation V386G was observed in all the sequences. Two isolates found with unique substitutions W339R and S375P are reported. The structural analysis of the wild type and mutant proteins revealed that the structural changes are accompanied by modification in the intraprotein interactions.

Anti-dengue activity of Andrographis paniculata extracts and quantification of dengue viral inhibition by SYBR green reverse transcription polymerase chain reaction.

BACKGROUND: Dengue virus is a leading cause of illness and death in the tropics and subtropics. As many as 400 million people are infected yearly. Dengue is caused by any one of four related viruses transmitted by mosquitoes. Currently, there is no vaccine to prevent infection with dengue virus and the most effective protective measures are those that avoid mosquito bites. When infected, early recognition and prompt supportive treatment can substantially lower the risk of medical complications and death. Nowadays, the search for natural plant products to fight against viral diseases has been increasing. AIMS AND OBJECTIVE: To test the anti-dengu viral activity of both ethanolic & aqueous extract of Andrographis paniculata. MATERIALS AND METHODS: In vitro antiviral activity were performed against dengue virus by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and SYBR green quantitative real-time polymerase chain reaction (PCR) method. Cytotoxicity was also evaluated by MTT. The dengue viral load (VL) inhibition in plant extracts was characterized by reverse transcription PCR (RT-PCR) analysis. RESULTS AND DISCUSSION: In this study, the maximum nontoxic dose (MNTD) of A. paniculata plant was determined by testing the ethanolic extracts against Vero cells in vitro. Antiviral assay based on cytopathic effects denoted by degree of inhibition upon treating DENV 1-4-infected Vero cells with MNTD of A. paniculata has the most antiviral inhibitory effects. These results were further verified with an in vitro inhibition assay using MTT and RT-PCR, in which 55%-97% of cell viability were recorded in DENV-1-4-infected cells in different duration. CONCLUSION: Ethanolic extracts treated with dengue VLs also showed a significant changes which were reflected in RT PCR assay.

Serological, molecular and clinical correlates of dengue from a tertiary care centre in Chennai, India.

Dengue disease is caused by dengue viruses 1-4 and has been ranked by the World Health Organisation (WHO) as the fastest spreading vector-borne viral disease. Dengue is often underreported and misdiagnosed due to a wide spectrum of clinical manifestations. Diagnosis of dengue is based on clinical case definitions and laboratory methods. Newer case definitions of dengue have been formulated by clinical studies in order to improve case detection. Owing to its epidemic potential, mortality and morbidity, there is a need for a rapid and accurate diagnostic assay for dengue in order to help the clinician in the early detection of cases and to prevent disease progression. A duplex real time PCR targeting the 3'UTR region for rapid and simultaneous detection of all dengue viruses serotypes (1-4) was standardized based on published literature. About 150 patients with acute undifferentiated febrile illness classified based on the 2009 WHO dengue case definition were tested using the duplex real time dengue PCR. Sequencing based PCR was performed on selected PCR positive samples for partial nucleotide sequence of the CprM gene and a phylogenetic tree was constructed. Statistical analysis was done using the MedCalc software. Out of the 126 patients classified as dengue disease positive, according to the 2009 WHO dengue case definition, 54% had "probable dengue", 43% had "dengue with warning signs" and 3% had "severe dengue". The performance of the duplex real time PCR was assessed among the various clinical groups of dengue and it was found that in the "dengue with warning signs group" PCR had a positive predictive value of 85.29% (range - 68.94% to 95.05%) when compared with dengue NS1 ELISA. The average time for PCR positivity was found to be four days from the onset of illness. The cycling threshold values obtained from real time PCR were used as a semi quantitative measure of viremia. Accordingly, there was a relatively low CT value among the "warning signs dengue group" when compared to the "probable dengue group". The use of the duplex PCR is suggested in the early diagnosis of dengue, especially in the 'warning signs' group of patients as they showed a higher positivity rate. Also, the use of the resultant CT value as a semi-quantitative measure of viremia will assist the clinician in early diagnosis and prevention of disease development.

Antiviral activity of Thiosemicarbazones derived from α-amino acids against Dengue virus.

The endemicity and seasonal outbreaks of Dengue disease in most tropical and subtropical countries underscores an urgent need to develop effective prevention and control measures. Development of a Dengue vaccine, which is complicated by the Antibody Dependent Enhancement effect (ADE), a viral inhibitor, seems prudent as it would inhibit the spread of the virus. In vitro methods such as MTT assay and plaque formation unit reduction assays were employed for screening the viral inhibitory property of α-amino acid based Thiosemicarbazides. The results elicits that at concentrations not exceeding the maximum non cytotoxic concentration (MNCC), these compounds completely prevented Dengue virus infection in vero cells as indicated by the absence of cytopathic effects in a dose-dependent manner. The high potency of Bz-Trp-TSC against all four types of Dengue virus infection elevates Thiosemicarbazide as a lead antiviral agent for Dengue disease. Screening small molecules for antiviral activity against the most rapidly spreading mosquito-borne viral disease is being explored by several research groups. Our findings would help to augment the efforts to identify the lead compounds for antiviral therapy to combat the Dengue disease. J. Med. Virol. 89:546-552, 2017. © 2016 Wiley Periodicals, Inc.

A novel indirect ELISA for diagnosis of dengue fever.

BACKGROUND & OBJECTIVES: Dengue fever (DF) is associated with significant morbidity and mortality in the tropical and sub-tropical regions of the world. Since there are no effective antiviral drugs for treatment, clinicians often rely on the accurate diagnosis of dengue fever to begin supportive therapy at early stages of the illness. The objective of this study was to develop an in-house dengue virus serotype 2 (DENV-2) non-structural protein- 5 (NS5) based indirect ELISA. METHODS: DENV-2 was raised in Vero cells and the viral proteins were separated and subsequently the NS5 protein was eluted. Serum samples from primary and secondary dengue fever patients; and acute and convalescent samples from Japanese encephalitis (JE) and West Nile virus (WNV) cases were used to validate the ELISA. RESULTS: The assay was found to be 100 per cent specific in detecting DENV-2 specific antibodies from patient's serum. However, in terms of sensitivity, the assay could detect IgM antibodies only from 90 per cent of the primary dengue samples. The IgM/IgG ratio of the primary and secondary samples was 7.24 and 0.64, respectively. INTERPRETATION & CONCLUSIONS: The results indicate that the DENV-2 NS5 ELISA is dengue group specific and can be used to differentiate dengue infection from other circulating Flavivirus infections. This NS5 ELISA can also be used to distinguish between primary and secondary dengue fever on the basis of IgM/IgG ratios. Further studies with larger sample sizes and different DENV serotypes are required to validate the ELISA.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon

Objective: To obtain luteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon (L.) (C. dactylon) Pers and evaluate the fraction's cytotoxicity and anti-Chikungunya potential using Vero cells. Methods: The ethanolic extract of C. dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus (CHIKV) fraction. Reverse phase-HPLC and GC-MS studies were carried out to identify the major phytochemicals in the fraction using phytochemical standards. Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells. Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) after treating the viral infected Vero cells with the fraction. Results: Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids, luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C. dactylon. The fraction was found to exhibit potent viral inhibitory activity (about 98%) at the concentration of 50 μg/mL as observed by reduction in cytopathic effect, and the cytotoxic concentration of the fraction was found to be 250 μg/mL. RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells. Conclusions: Luteolin and apigenin rich ethanolic fraction from C. dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon

OBJECTIVE@#To obtain luteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon (L.) (C. dactylon) Pers and evaluate the fraction's cytotoxicity and anti-Chikungunya potential using Vero cells.@*METHODS@#The ethanolic extract of C. dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus (CHIKV) fraction. Reverse phase-HPLC and GC-MS studies were carried out to identify the major phytochemicals in the fraction using phytochemical standards. Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells. Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) after treating the viral infected Vero cells with the fraction.@*RESULTS@#Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids, luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C. dactylon. The fraction was found to exhibit potent viral inhibitory activity (about 98%) at the concentration of 50 µg/mL as observed by reduction in cytopathic effect, and the cytotoxic concentration of the fraction was found to be 250 µg/mL. RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells.@*CONCLUSIONS@#Luteolin and apigenin rich ethanolic fraction from C. dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus.

Development and evaluation of flavi-immunoglobulin M capture enzyme-linked immunosorbent assay.

In this study, we report the evaluation of In-house flavi virus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA), which can be used as a screening test to determine the infecting flavivirus serotype over the current serological methods. A panel of 88 sera (inclusive of well characterized dengue, Japanese Encephalitis (JE) and West Nile virus (WNV) positive and negative samples tested and confirmed by commercial kit) was used for evaluation of the kit. The sensitivity and specificity of the In-house capture assay versus the commercial kit for the sero-diagnosis of dengue was 100% and 87% respectively, for JE IgM, it was found to be 90% and 100% respectively, and for West Nile it was 87.5% and 90.9%. Based on the study, we concluded that this flavivirus-serotyping ELISA provides rapid results and may be used as an accurate alternate to other serological tests for the specific diagnosis of flavivirus infections.

Molecular characterization of wild-type measles viruses in Tamil Nadu, India, during 2005-2006: relationship of genotype D8 strains from Tamil Nadu to global strains.

Molecular characterization of measles viruses is a valuable tool for measuring the effectiveness of measles control and elimination programmes. WHO recommends that virological surveillance be conducted during all phases of measles control to document circulation of indigenous strains and trace future importation. This report describes the genetic characterization of wild type measles viruses from Tamil Nadu, India isolated between January 2005 and January 2006. In the study, 304 suspected measles cases (292 from 56 outbreaks and 12 sporadic cases) were investigated. Blood samples were collected from suspected measles outbreaks and 11 suspected sporadic cases and tested for the presence of measles and rubella specific IgM. Based on serological results, 53 outbreaks were confirmed as measles, 2 as a combination of measles and rubella, and 1 negative for both. Eight sporadic cases were confirmed as measles and one as rubella. Throat swab and urine samples were collected for virus isolation and 28 isolates were obtained. Sequencing and analysis showed that 3 isolates belonged to genotype D4 and 25 to genotype D8. Comparison of the genotype D8 sequences from Tamil Nadu with previously reported genotype D8 sequences from India and abroad showed six distinct clusters with Tamil Nadu strains forming two clusters. This study has established baseline molecular data and is the first report that describes genetic diversity of circulating measles strains in Tamil Nadu, a state in India. D8 has multiple lineages and this has been linked with importation of measles into the USA and UK.

Cytotoxicity and degree of conversion of orthodontic adhesives.

OBJECTIVES: To test the hypothesis that there is no difference in the cytotoxicity related to the modes of polymerization of five commercially available orthodontic bonding resins, with and without an oxygen-inhibited layer (OIL), and to evaluate the degree of conversion (DC) of these resins and correlate this to cytotoxicity. MATERIALS AND METHODS: Five commercially available orthodontic bonding resins were tested for cytotoxicity and DC. Thirty-six disks of standardized dimensions, for each resin, were used for cytotoxicity assessment. Half of them were washed with 99% acetone to remove the OIL (washed resins), and the remaining disks were left intact (intact resins). Glass disks were used as a control. Vero cells were exposed to intact and washed resins on day 1. Cell viability was determined by tetrazolium bromide reduction assay 1, 3, and 6 days after exposure. The DC of the adhesive specimens of each resin, prepared with a procedure identical to the clinical bonding process, was assessed by Fourier transform infrared spectroscopy. RESULTS: Single-cured systems were comparatively less cytotoxic than dual-cured systems. With removal of the OIL, increased cell viability was noted only with two resins on all three days. Resins tested showed differences in DC. A positive correlation was demonstrated by two resins. CONCLUSION: The hypothesis is rejected. Single-cured systems are superior to dual-cured systems in exhibiting comparatively less toxicity and higher DC. A significant positive correlation was not established between cytotoxicity and DC.