Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

<p>Abstract</p> <p>Background</p> <p>Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported.</p> <p>Results</p> <p>An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever) and alphavirus (Chikungunya). The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation <it>vis a vis </it>conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only.</p> <p>Conclusion</p> <p>These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.</p

Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956–2007

<p>Abstract</p> <p>Background</p> <p>Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001–2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis.</p> <p>Results</p> <p>The region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I → T, A → T and L → S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997–98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1–4) showing proximity to isolates from diverse geographic locations.</p> <p>Conclusion</p> <p>The extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India.</p

Transcriptomic profile of host response in Japanese encephalitis virus infection

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy with the highest mortality rate of 30-50%. The purpose of this study was to understand complex biological processes of host response during the progression of the disease. Virus was subcutaneously administered in mice and brain was used for whole genome expression profiling by cDNA microarray.</p> <p>Results</p> <p>The comparison between viral replication efficiency and disease progression confirms the active role of host response in immunopathology and disease severity. The histopathological analysis confirms the severe damage in the brain in a time dependent manner. Interestingly, the transcription profile reveals significant and differential expression of various pattern recognition receptors, chemotactic genes and the activation of inflammasome. The increased leukocyte infiltration and aggravated CNS inflammation may be the cause of disease severity.</p> <p>Conclusion</p> <p>This is the first report that provides a detailed picture of the host transcriptional response in a natural route of exposure and opens up new avenues for potential therapeutic and prophylactic strategies against Japanese encephalitis virus.</p

Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat

Difference in somatic embryogenetic ability of cultured leaf explants of four genotypes of Solanum melongena L

High frequency of somatic embryogenesis was obtained from leaf explants of 4 genotypes of Solanum melongena L. Varietal response was noticed with reference to optimum concentration of NAA required for somatic embryogenesis. Percent explants forming somatic embryos varied from 15.2-90.3, mean number of plants regenerated/explant from 1.5-29.5. In terms of their embryogenic competence and plant regeneration the genotypes could be rated in decreasing order as F1 hybrid Suphal followed by PPL, DRLS and Kalalong. Initital inoculum size had a distinct correlation with number of plants regenerated. Decline in embryogenic competence was observed in all 4 genotypes under long-term culture. Plantlets regenerated from somatic embryos were established on soil and grown to maturity.Embryogenèse somatique d'explants de feuilles de quatre génotypes de Solanum melongena. Des explants foliaires de 4 génotypes de Solanum melongena L donnèrent lieu, avec une fréquence importante, à de l'embryogenèse somatique. Les réponses variétales furent notées, en corrélation avec la concentration d'acide a naphtylacétique (ANA) nécessaire pour l'embryogenèse somatique. La proportion d'embryons somatiques formés varia de 15,2 à 90,3 pour 100 embryons et le nombre moyen de plantes régénérées par embryon de 1,5 à 29,5. On peut classer les génotypes en fonction de leur compétence pour l'embryogenèse et la régénération de plantes de la façon suivante : hybride F1 Sulphal, PPL, DRLS, Kalalong, par ordre décroissant. Le nombre de plantes régénérées était en rapport direct avec la taille initiale de l'inoculum. Un déclin de la compétence à l'embryogenèse fut observé par les 4 génotypes sur les cultures âgées. Les plantules régénérées à partir d'embryons somatiques furent cultivées sur sol, jusqu à maturité

Comparison of a dipstick enzyme-linked immunosorbent assay with commercial assays for detection of Japanese encephalitis virus-specific IgM antibodies

Background: Japanese encephalitis (JE) is a major public health concern in Asia including India. Objectives: To evaluate an in-house developed dipstick enzyme-linked immunosorbent assay (ELISA) test vis-à-vis two commercial kits for detection of JE virus-specific IgM antibodies. Setting and Design: Comparative study carried out in Research and Development centre. Materials and Methods: A total of 136 specimens comprising 84 serum and 52 CSF samples were tested by in-house dipstick ELISA, Pan-Bio IgM capture ELISA (Pan-Bio, Australia) and JEV CheX IgM capture ELISA (XCyton, India). Results: The overall agreement among all three tests was found to be 92% with both serum and cerebrospinal fluid (CSF) samples. The sensitivity of the dipstick ELISA was found to be 91% with serum and 89% with CSF samples respectively. The specificity of the dipstick ELISA with reference to both commercial assays was found to be 100% in serum and CSF samples in this study. Conclusions: The in-house dipstick ELISA with its comparable sensitivity and specificity can be used as a promising test in field conditions since it is simple, rapid and requires no specialized equipment

Fusogenic peptide as diagnostic marker for detection of flaviviruses

Background: Dengue, Japanese encephalitis, West Nile encephalitis, yellow fever are the common flaviviral diseases associated with high morbidity and mortality. The initial symptoms of most of the flaviviral infections are similar to each other as well as to some other viral diseases. Making clinical diagnosis, therefore, becomes a challenging task for the clinician. Several studies have been reported on using detection of serum antibodies against flavivirus for the diagnosis of specific flaviviral disease; no field-based pan-flavi virus detection system is available, which can be used in low-endemicity areas for differentiation of flaviviral disease from other viral diseases. Aim: To identify a conserved amino acid sequence among all flaviviruses and evaluate the antibody formed against the conserved peptide to develop pan-flavivirus detection system. Materials and Methods: In the present study we have compared amino acid sequences of several flaviviruses and identified a conserved amino acid sequence lying in domain II of envelope protein. Results : A peptide having the conserved amino acid sequence was used to generate polyclonal antibodies and these antibodies were used to detect several flaviviruses. Anti-peptide polyclonal antibodies selectively recognized flaviviruses and did not detect non-flaviviruses. Anti-peptide antibodies detected presence of virus in serum spiked with pure virus preparations. Conclusion: The study offers a rationale for development of pan-flavivirus capture assay suitable for low endemic areas