Lineage shift of dengue virus in Eastern India: an increased implication for DHF/DSS.

Dengue fever, a mosquito-borne viral disease, has become a major public health problem with marked expansion in recent decades. Dengue has now become hyperendemic in India with co-circulation of all the four serotypes. Herein, we report an unprecedented outbreak which occurred during August to October 2011 in Odisha, eastern India. This is the first report of a large epidemic in Odisha. Detailed serological and molecular investigation was carried out to identify the aetiology. Almost half of the samples were found to be dengue antigen (NS1) positive. Further molecular assays revealed circulation of mixed dengue serotypes (DENV-2 and DENV-3). Cosmopolitan genotype of DENV-2 and -3 were identified as the aetiology by phylogenetic analysis. Interestingly, a new lineage of DENV-3 within cosmopolitan genotype was incriminated in this outbreak. The emergence of the unprecedented magnitude of the dengue outbreak with the involvement of a novel lineage of DENV in a newer state of India is a major cause for concern. There is an urgent need to monitor phylodynamics of dengue viruses in other endemic areas.

Unusual and rare manifestations of dengue during a dengue outbreak in a tertiary care hospital in South India.

Dengue is the most rapidly spreading mosquito-borne viral disease in the world, and as a larger proportion of the population is being affected, more unusual manifestations are being reported. Very few studies have documented unusual manifestations of dengue in South India. This prospective study was undertaken from July 2011 to June 2013 to document rare manifestations of dengue fever in 175 hospitalized patients. The clinical diagnosis was confirmed by the detection of NS1Ag, dengue IgM, or IgG by ELISA and/or a RT-PCR and CDC real-time PCR for dengue virus (DENV) RNA. The daily profiles of the hematological and biochemical investigations were followed and recorded. Unusual and rare manifestations of dengue were documented for 115 patients (66 %). Hepatitis was observed in 70 % of the cases. Pleural effusion was seen in 11 %, acute renal failure in 10 %, neurological complications such as encephalitis in 7.4 %, myocarditis in 9 %, and bleeding gastric ulcers in 3.4 % of the cases. DENV serotype 2 was more prevalent in patients with unusual manifestations of dengue in our study. The WHO classification system does not include unusual and rare manifestations; hence, it is essential to be aware of these manifestations and closely monitor them for better clinical management and outcome of patients.

Analysis of antibody response (IgM, IgG, IgG3) to Chikungunya virus using panel of peptides derived from envelope protein for serodiagnosis.

BACKGROUND: Many epidemic outbreaks of Chikungunya fever (CHIKF) have been reported throughout the world including India after its reemergence in 2005. The immuno protective role of envelope proteins during Chikungunya virus (CHIKV) infection has been reported. With the aim of identifying the immunodominant epitopes within the envelope protein we investigated the detailed analysis of fine specificity of antibody response in different individuals during CHIKV infection. METHODS: The peptides corresponding to the full length of E1, E2 and E3 proteins of S27 strain of CHIKV were synthesized and their seroreactivity with CHIKV positive patients' sera collected from different epidemic regions of India was determined using indirect ELISA. RESULTS: The data analysis reveals many potent epitopes throughout the length of envelope E2 protein thus displaying it as the most promising antigen for diagnostic purpose. We found that the main IgG isotype response to envelope protein was predominantly of subclass IgG3. Interestingly, most of the epitopes were found to be conserved for detecting IgM, IgG and IgG3 antibody response. CONCLUSIONS: Peptides E2P3, E2P7, E2P16 and E2P17 were revealed as the most immunodominant peptides that together can form the basis for designing an accurate, economical and easy to synthesize a peptide-based immunodiagnostic for CHIKV. This study provides new and important insight into the humoral response generated by CHIKV S27 strain during the early phase of infection.

Comparative complete genome analysis of dengue virus type 3 circulating in India between 2003 and 2008.

Dengue is endemic in most parts of the tropics including India. So far, complete genome information for Indian dengue isolates is not available. In the present study, we characterized the genome of three dengue type 3 viruses isolated from India. The genomes of all three viruses were found to be 10,707 bp long with an ORF encoding 3390 aa. Extensive molecular phylogenetic analysis based on comparison of the complete genome and envelope gene classified the recent Indian viruses into genotype III (lineage III), revealing a shift of lineage from lineage V. The sequence analysis revealed several non-conservative changes in major structural proteins. This study clearly indicates that the genotype III (lineage III) dengue type 3 viruses have been continuously circulating in major parts of India since 2003 and are responsible for the recent major outbreaks all over India. This is the first extensive study on complete genome analysis of dengue type 3 viruses in India.

Emergence of dengue virus type 4 (genotype I) in India.

Dengue is an emerging arboviral disease and currently poses the greatest arboviral threat to human health. In recent decades, there has been a substantial increase in dengue outbreaks in many parts of the world including India. We performed an in-depth investigation of a major dengue outbreak in Andhra Pradesh, southern India in 2007 by serology, virus isolation, RT-PCR and genotyping. The results revealed an unusual emergence of dengue virus type 4 (DENV-4) along with the prevailing DENV-3. Phylogenetic analysis based on complete envelope gene of 182 globally diverse DENV-4 isolates demonstrated the involvement of a unique clade of genotype I of DENV-4 in the outbreak. This study also demonstrated a clear shift in the dominant serotype from DENV-3 to DENV-4 in India. This is the first report regarding the molecular characterization of Indian isolates of DENV-4, which has the potential to be involved in future outbreaks.

Clinical features and molecular diagnosis of Chikungunya fever from South India.

An epidemic of Chikungunya fever of unprecedented magnitude occurred in many parts of India in early 2006 after an interval of 33 years, and there has been a resurgence in some parts of South India since June 2007. The article highlights clinical manifestations of infection and various molecular tests that were used for diagnoses of Chikungunya virus infection. Of particular interest is the real-time loop-mediated isothermal amplification (RT LAMP) assay, which is rapid and cost-effective and can be adopted at ill-equipped laboratories. Clinical symptoms were characterized by a triad of fever, rash, and severe rheumatic manifestations. RT LAMP identified 20 additional Chikungunya virus-positive cases, compared with reverse-transcriptase polymerase chain reaction. Chikungunya virus was isolated from 20 randomly selected samples. Genotyping of the virus isolates revealed that the East Central South African genotype of Chikungunya virus was the etiologic agent of this epidemic. Molecular diagnosis is an important tool to identify such new vectorborne viral illnesses.

Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates.

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented explosive epidemic after a gap of 32 years in India is a point of major public health concern. In 2007 again there was outbreak in Kerala, India, affecting more than 25,000 cases with many reported mortalities. To understand the molecular basis of this high virulence and its implication in large-scale epidemic, a detailed systematic serological, virological and molecular investigation was undertaken with the epidemic samples of Kerala-2007. The comparative analysis of full genome sequence of Chikungunya virus isolate of 2007 with 2006 revealed three unique substitutions in structural and non-structural genes of 2007 isolate [two in E1 region (V14A and A226V) and one in Nsp1 (M184T)]. Our finding further substantiates the association of A226V shift in E1 gene with evolutionary success possibly due to adaptation in the mosquito vector with progression of epidemic, as observed in Reunion Island. This A226V shift which was absent in all 2006 Indian isolates, is found to be present in the four 2007 isolates, analysed in this study. These unique molecular features of the 2007 isolates with the progression of the epidemic from 2005 to 2007 demonstrate their high evolutionary and epidemic potential and thereby suggesting possible implication in higher magnitude and virulence of this outbreak.

Rapid and real-time detection technologies for emerging viruses of biomedical importance.

The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing, and/or detection of antibodies,which are not very sensitive and specific. The recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. We have developed two real-time assays ie., SYBR green I based real time reverse transcription polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (LAMP) assay for rapid detection as well as typing of some of the emerging viruses of biomedical importance viz. dengue, Japanese encephalitis, chikungunya, west Nile, severe acute respiratory syndrome virus (SARS) etc. Both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity, specificity. One of the most important advantages of LAMP is its field applicability, without requirement of any sophisticated equipments. Both these assays have been extensively evaluated and validated with clinical samples of recent epidemics from different parts of India. The establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future.

Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei.

Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 103  CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings.

Simultaneous detection and serotyping of dengue infection using single tube multiplex CDC Dengue Real-Time RT-PCR from India.

Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well serotyping assay in near future for rapid identification of dengue suspected sample screening.

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus.

The development of a one-step SYBR Green I-based real-time RT-PCR assay is reported for detection and quantification of Chikungunya virus (CHIKV) in acute-phase patient serum samples by targeting the E1 structural gene. A linear relationship was obtained between the virus concentration and cycle threshold (C(t)) value over a range of 10(7)-0.1PFU/ml. The reported assay was found to be 10-fold more sensitive compared to conventional RT-PCR with a detection limit of 0.1PFU/ml. The feasibility of this reported assay system for clinical diagnosis was validated with 51 suspected acute-phase serum samples of the recent CHIKV epidemic in southern India, 2006. The comparative evaluation with acute-phase patient serum samples revealed the higher sensitivity of real-time RT-PCR assay by picking up six additional samples with low copy number of template. None of the healthy serum samples analyzed in this study showed amplification. The quantification of the viral load in the acute-phase serum samples was also determined employing the standard curve, which varies from 0.1 to 10(7)PFU/ml. These findings demonstrated that the reported assay has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of Chikungunya virus in acute-phase patient serum samples.

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus.

One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold (C(t)) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples.

East Central South African genotype as the causative agent in reemergence of Chikungunya outbreak in India.

Chikungunya fever is an important arboviral infection prevalent throughout Africa and Southeast Asia. Recently, in 2006, it has reemerged in many parts of India, affecting more than a million persons. A detail serological, virological, and molecular investigation of this unprecedented outbreak was carried out by collecting and studying 540 samples from all the affected regions of India during this epidemic. An in-depth investigation revealed the presence of anti-Chikungunya antibodies in 68% of the samples and genomic RNA in 49% of them. In addition 32 Chikungunya viruses were isolated from 45 representative polymerase chain reaction-positive samples. The nucleotide sequences of partial E1 gene of 25 representative Chikungunya viruses were deciphered. The sequence analysis indicated that all the isolates of this epidemic belonged to the new Indian Ocean island clade of East Central South (ECS) African genotype. This study conclusively proved the genotype shift from Asian to ECS African as the major factor in the reemergence of Chikungunya in an unprecedented outbreak in India after a gap of 32 years.

Comparison of a dipstick dot-ELISA with commercial assays for anti-dengue virus IgM antibodies.

Dengue virus infections have undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Dengue virus causes life-threatening complications characterized by dengue hemorrhagic fever and dengue shock syndrome. No standard validated test systems are available for serological diagnosis of dengue virus infection. This creates problems in the diagnosis and proper management of patients. Evaluation of a Defense Research and Development Establishment (DRDE) dengue virus dipstick dot-ELISA test, developed in house, vis-à-vis commercially available immunodiagnostic kits was carried out for detection of IgM antibodies. The DRDE dengue dipstick dot-ELISA was performed on the basis of the dot-ELISA principle. Commercial tests, namely, the Panbio Dengue IgM Capture ELISA system (Panbio, Sinnamon Park, Australia) and Pathozyme Dengue M (Omega Diagnostics, Alva, UK), were performed according to the manufacturers' protocols. Ninety-one serum samples collected from the states of Kerala and Delhi, India during August and November of 2004 were used in the present study. The overall agreement among all three tests was found to be only 72.16% for IgM antibodies. Correlations between the DRDE dipstick dot-ELISA and the Panbio kit, between the DRDE dipstick dot-ELISA and the Pathozyme Dengue M kit, and between the Panbio kit and the Pathozyme Dengue M kit were found to be 96, 93, and 94%, respectively. Although the Panbio kit is widely used in various laboratories in India, the DRDE dipstick dot-ELISA promises to be a useful kit because of its field applicability and comparable sensitivity.

Evaluation of multiple antigenic peptides based on the Chikungunya E2 protein for improved serological diagnosis of infection.

In recent years, Chikungunya virus (CHIKV) reemerged and numerous outbreaks were reported all over the world. After screening CHIKV-positive sera, we had already reported many dominant epitopes within the envelope E2 protein of CHIKV. In the present study, we aimed at developing a highly sensitive immunodiagnostic assay for CHIKV based on a multiple antigenic peptide (MAP) approach using selective epitopes of the E2 protein. MAPs in four different E2 peptide combinations were screened with CHIKV-positive sera. The MAPs reacted with all CHIKV-positive sera and no reactivity was seen with healthy or dengue-positive sera. Our results indicate that MAP 1 seems to be an alternate antigen to full-length protein E2 for immunodiagnosis of CHIKV infections with high sensitivity and specificity.

Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India.

Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India.

Serological & virological investigation of an outbreak of dengue fever in Gwalior, India.

BACKGROUND & OBJECTIVES: An outbreak of febrile illness occurred between September to November 2001 in Gwalior, Madhya Pradesh affecting individuals mostly in the age group < 30 yr. A total of 312 febrile indoor patients suspected to have dengue infection were investigated. METHODS: The investigation included examination of blood samples from patients for dengue specific IgM and IgG antibodies, isolation of virus in suckling mouse pups and in C(6/36) cell line followed by confirmation and typing through reverse transcriptase-PCR and nested PCR. RESULTS: The serological analysis of the 312 samples indicated 65 per cent positivity of which 21 per cent are of recent infection as indicated by the presence of IgM antibody and 78 per cent are found to be secondary in nature by showing the presence of IgG and/or IgM antibodies. The RT-PCR analysis of patients' sera employing dengue virus group specific conserved amplimer confirmed the etiological agent as dengue complex by showing the characteristic 511 bp amplicons. None of the antibody positive samples were found to be positive by RT-PCR. A total of 13 (6%) samples positive by RT-PCR, were processed for virus isolation in mouse pups and in C(6/36) cells. Of these 9 samples (80%) were confirmed positive for virus isolation as identified by RT-PCR. INTERPRETATION & CONCLUSION: The typing of isolates by nested PCR employing serotype specific amplimer revealed 119 bp amplicon characteristic of dengue virus type-2 and thus confirming the outbreak attributed to dengue virus type-2.

Inhibitory potential of neem (Azadirachta indica Juss) leaves on dengue virus type-2 replication.

In the present study we report in vitro and in vivo inhibitory potential of crude aqueous extract of neem leaves and pure neem compound (Azadirachtin) on the replication of Dengue virus type-2. In vitro antiviral activity of aqueous neem leaves extract assessed in C(6/36) (cloned cells of larvae of Aedes albopictus) cells employing virus inhibition assay showed inhibition in dose dependent manner. The aqueous extract of neem leaves at its maximum non-toxic concentration of 1.897 mg/ml completely inhibited 100-10,000 TCID(50) of virus as indicated by the absence of cytopathic effects. The in vivo protection studies with neem leaves extract at its maximum non-toxic concentrations 120-30 mg/ml resulted in inhibition of the virus replication as confirmed by the absence of Dengue related clinical symptoms in suckling mice and absence of virus specific 511 bp amplicon in RT-PCR. The pure neem i.e. Azadirachtin did not reveal any inhibition on Dengue virus type-2 replication in both in vitro and in vivo systems.

Anti-microbial activity of a new vaginal contraceptive NIM-76 from neem oil (Azadirachta indica).

Efficacy of NIM-76, a spermicidal fraction from neem oil, was investigated for its antimicrobial action against certain bacteria, fungi and Polio virus as compared to whole neem oil. The NIM-76 preparation showed stronger anti-microbial activity than the whole neem oil. It inhibited growth of various pathogens tested including Escherichia coli and Kleibsiella pneumoniae which were not affected by the whole neem oil. NIM-76 also exhibited antifungal activity against Candida albicans and antiviral activity against Polio virus replication in vero cell lines. It also protected mice from systemic candidiasis as revealed by enhanced % survival and reduced colony forming units of C. albicans in various tissues. This shows that NIM-76 has a potent broad spectrum anti-microbial activity.

Freshwater cyanobacterium Microcystis aeruginosa (UTEX 2385) induced DNA damage in vivo and in vitro.

Microcystins are a family of potent hepatotoxins and liver tumor promoters produced by several genera of cyanobacteria including Microcystis, Nodularia, Anabena, Nostoc, etc. They are chemically very stable and represent a public health threat when they occur in water used for human consumption. We investigated the DNA damage effects of M. aeruginosa UTEX 2385 in mouse liver in vivo and also in mammalian cells in vitro. The DNA damage effect is compared with purified toxin microcystin-LR (MCLR) in non-hepatic cells viz. baby hamster kidney cells (BHK-21) and mouse embryo fibroblasts primary cells (MEF). Cell-free extracts of UTEX 2385 induced significant DNA fragmentation at 0.5, 1 and 2 LD(50) (32.7, 65.4 and 130.8 mg/kg, respectively) and it was also time dependent. M. aeruginosa UTEX 2385 and MCLR induced significant DNA fragmentation in BHK-21 and MEF cells at 100 and 1.0 µg/ml concentration. Electrophoretic analysis revealed necrotic DNA damage by UTEX 2385 in vivo. Both the toxins caused smear in agarose gel electrophoresis indicating the necrotic DNA damage in MEF cells, whereas, multiple DNA fragments in BHK-21 cells. The DNA damage effect of the toxin is supported by data on hepatotoxicity in vivo and cytotoxicity in vitro.