Rajani Malla

Re-emergence of dengue virus serotype 2 strains in the 2013 outbreak in Nepal

Background & objectives: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. Methods: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. Results: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. Interpretations & conclusions: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region.

Similarity Between Piriformospora indica and Sebacina vermifera Sensu Members of the Order Sebacinales Based on Immunological Techniques

The acid phosphatases in Piriformospora indica and Sebacina vermifera sensu were similar in their molecular mass. The raised antibody against acid phosphatase (ACPase) of P. indica showed maximum ELISA reading with S. vermifera sensu supporting strong relationship between these two fungi. The immunoblot analysis showed the strong reactivity of antiserum with S. vermifera sensu. The antiserum blotted the bands of 66 kDa in S. vermifera separated in denatured—PAGE at equidistance with P. indica. The enzyme assay zymogram of non-denatured-ACPase showed parallel bands at the same height in native PAGE. The antiserum also localized the enzyme in S. vermifera by immunofluorescence technique, showing strong relationship of this fungus with P. indica. The immunogold labeling of antiserum from P. indica precisely localized the enzyme in cytoplasm and vacuoles of S. vermifera supporting the strong immunological link between these two fungi. Two-dimensional map of crude protein of these two fungi showed some differences in minor proteins.

Molecular Techniques to Study Polymorphism between Closely Related Microorganisms in Relation to Specific Protein Phosphatase

Phylogenetically, the Sebacinaceae that include P. indica recognized a new order, the Sebacinales. The order primarily contains the genera Sebacina, Tremelloscypha, Efibulobasidium, Craterocolla, and Piriformospora. In this study, attempts were made to purify acid phosphatase from P. indica. The fungus produced only one form of intracellular acid phosphatase irrespective of the phosphate concentration. The enzyme was possibly a constitutive enzyme showing molecular mass of 66 kD as separated by SDS-PAGE. The enzyme showed the pH and temperature optima of 5.3 and 40°C, respectively. K m for p-NPP (monoester) was 0.35 mM. Antibodies raised against cytosolic acid phosphatase using gel band in Native PAGE after selective precipitation with ammonium sulfate followed by gel filtration and ion exchange chromatography gave sufficient quantities of antibodies based on immunoblot analysis.