siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus.

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58% and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80% reduction in viral plaque counts and 94% inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68–73% and 87–88% reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

In silico approach to evaluate evolutionary relatedness of significant Lung cancer proteins.

Lung cancer is feared to be deadly and is caused due to the involvement of a cascade of proteins significant among which are EPAS1, Thrombomodulin, Metallothionein and Matrix Metallo Proteinase. The evolutionary aspects of these proteins are important to study the extent of conservation and mutation across different generations in diverse species. The rooted and unrooted tree would depict the relatedness among organisms and the ancestor of the organisms involved in the study. The evolutionary histories of groups of species are one of the most widely used tools throughout the life sciences, as well as objects of research with in systematic, evolutionary biology. In every phylogenetic analysis reconstruction produces trees. These trees represent the evolutionary histories of many groups of organisms, bacteria due to horizontal gene transfer and plants due to process of hybridization. Through comparison with several species of healthy organism, one may determine where the defective mutation is located, and then determine how to treat the disease. This strategy may help us to identify the mutations that had occurred in evolutionary conserved residues. We feel this method can be useful for understanding evolutionary rate variation, and for understanding selection variation on different characters. The various tree topologies along with their significant homolog’s would enable a thorough understanding of the disease.

Diversity of Arbuscular Mycorrhizal Fungi in Oak-Pine Forests and Agricultural Land Prevalent in the Kumaon Himalayan Hills, Uttarakhand, India.

The diversity and abundance of arbuscular mycorrhizal fungi (AMF) was assessed in the Kumaon Himalayan foothills. Four typical ecosystems were selected in the Sat-Tal area located in the Nainital, district in Uttarakhand, India, representing vegetation change due to human settlement and selective logging of native oak. Besides a natural oak forest, a mixed pine-oak forest, a pure pine stand and an arable field were sampled. The latter was cropped with black gram (Vigna mungo L. Hepper) and maize (Zea mays L.) during the rainy season from June to September and rotated with wheat (Triticum aestivum L.) during winter for the last 10 years. Only cow dung compost used for fertilization. The highest AMF spore abundance throughout the year was recorded in soil samples from the pine-oak mixed forest, followed by the pine and oak forests and the agricultural field. At all sites, the lowest spore abundance was recorded at the end of the winter season in March, and the highest in October after the rainy season. Whereas in October, Glomus claroideum, Acaulospora scrobiculata and A. spinosa were found at all sites, in March it was only G. intraradices which occurred everywhere. The highest AMF spore morphotype richness was recorded in samples from the oak forest. In AMF-trap cultures set up with field soil inocula, the dominant species recovered were G. intraradices, G. etunicatum and A. scrobiculata. As compared to the field samples, trap culturing of one year enhanced spore abundance but entailed a loss of AMF richness. The study revealed differences in AMF community composition and structure among sites characterized by different land use systems.

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (Npro) protein of bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (Npro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV Npro–His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the Npro protein was confi rmed by western blot using anti- BVDV hyperimmune cattle, sheep and goat serum, and anti-Npro rabbit serum. When rabbits were immunized with the Npro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite Npro-specifi c antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the fi eld cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for Npro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV Npro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.

Community practices of using bed nets & acceptance & prospects of scaling up insecticide treated nets in north-east India.

BACKGROUND & OBJECTIVE: The National Vector Borne Disease Control Programme (NVBDCP), India has been promoting and scaling up the use of insecticide-treated nets (ITNs) in the tribal dominated malarious areas of north-east India. But, information on sleeping habits, bed net ownership and use practices, and feedback of communities in ITN--targeted areas is needed for formulating a strategic framework for upscaling the coverage of ITNs. We carried out a community-based cross-sectional survey in select areas of Nagaland and Mizoram (where ITNs were introduced) along with Assam (where ITNs were not introduced) to know the response of community. METHODS: Following large scale introduction of ITNs during 2001-2002 in the north-eastern States of Nagaland and Mizoram by NVBDCP, India, a cross-sectional community-based survey was undertaken in April-May, 2003 covering 435 households of Nagaland and 464 households in Mizoram, using a structured questionnaire, to assess the demographic variables relevant to bed net use, bed net washing practices and acceptability of ITNs etc., for upscaling the coverage of ITNs in the surveyed communities. A total of 448 households in 8 villages in a non-ITN PHC area of Assam were served as the control area. RESULTS: The average bet net usage per family (2.01 to 2.65). Bed net use was mostly seasonal in Nagaland where a majority (65.1-78.7%) used bed nets only during summers and monsoon as compared to the year round use in Mizoram (83.5%) and Assam (78.9%). Frequent washing of nets was most common in Assamese communities with 77 per cent households washing their nets at least once in a month. More than two third users favoured use of ITNs over the conventional indoor residual spray of DDT for malaria control. INTERPRETATION & CONCLUSION: ITNs are widely acceptable in the user communities and a demand for ITNs is evident in the non-user communities of north-east India. Approach of treating community owned nets through an efficient service delivery mechanism will be a viable option for upscaling the ITN coverage.

Community practices of using bed nets & acceptance & prospects of scaling up insecticide treated nets in north-east India.

Background & objectives: The National Vector Borne Disease Control Programme (NVBDCP), India has been promoting and scaling up the use of insecticide-treated nets (ITNs) in the tribal dominated malarious areas of north-east India. But, information on sleeping habits, bed net ownership and use practices, and feedback of communities in ITN - targeted areas is needed for formulating a strategic framework for upscaling the coverage of ITNs. We carried out a community-based cross-sectional survey in select areas of Nagaland and Mizoram (where ITNs were introduced) along with Assam (where ITNs were not introduced) to know the response of community. Methods: Following large scale introduction of ITNs during 2001-2002 in the north-eastern States of Nagaland and Mizoram by NVBDCP, India, a cross-sectional community-based survey was undertaken in April-May, 2003 covering 435 households of Nagaland and 464 households in Mizoram, using a structured questionnaire, to assess the demographic variables relevant to bed net use, bed net washing practices and acceptability of ITNs etc., for upscaling the coverage of ITNs in the surveyed communities. A total of 448 households in 8 villages in a non-ITN PHC area of Assam were served as the control area. Results: The average bet net usage per family (2.01 to 2.65). Bed net use was mostly seasonal in Nagaland where a majority (65.1-78.7%) used bed nets only during summers and monsoon as compared to the year round use in Mizoram (83.5%) and Assam (78.9%). Frequent washing of nets was most common in Assamese communities with 77 per cent households washing their nets at least once in a month. More than two third users favoured use of ITNs over the conventional indoor residual spray of DDT for malaria control. Interpretation & conclusion: ITNs are widely acceptable in the user communities and a demand for ITNs is evident in the non-user communities of north-east India. Approach of treating community owned nets through an efficient service delivery mechanism will be a viable option for upscaling the ITN coverage.

Detection & molecular confirmation of a focus of Plasmodium malariae in Arunachal Pradesh, India.

BACKGROUND & OBJECTIVE: During a malaria epidemiological study in Arunachal Pradesh, Plasmodium malariae like human malaria parasites were seen in blood smears from fever cases. The study was undertaken to detect the presence of P. malariae and to confirm its identity through DNA based polymerase chain reaction approach. METHODS: Fever survey was carried out in 22 villages in Indo-Myanmar bordering district of Lohit, Arunachal Pradesh in 2005. Morphologically suspected P. malariae cases were confirmed using nested PCR based on 18S small subunit ribosomal DNA gene sequence. RESULTS: Screening of 1,995 fever cases resulted in 9 probable cases of P. malariae based on morphological identification in Chakma tribe people residing in 2 villages. Nested PCR confirmed the identity of all probable cases of P. malariae by producing diagnostic band of 144 bp. PCR method was able to detect mixed infection of P. malariae with P. vivax and with P. falciparum. INTERPRETATION & CONCLUSION: P. malariae may have been present in Arunachal Pradesh but most probably is being misdiagnosed due to its close resemblance with P. vivax, especially in ring forms. Estimation of actual case load of P. malariae in north-east India is, therefore, important with accurate species identification using molecular methods.

Characterization of ITS2 rDNA of Anopheles philippinensis and Anopheles nivipes (Diptera: Culicidae) from north-east India.

Anopheles (Cellia) philippinensis Ludlow and Anopheles (Cellia) nivipes (Theobald) are two closely related, morphologically very similar, mosquito species in the Annularis group, which play a supportive role in malaria transmission in north-east India. We amplified and performed sequence analysis for the internal transcribed spacer 2 (ITS2) locus of ribosomal DNA (rDNA) gene of morphologically confirmed specimens of these two species from the states of Assam and Nagaland. An. philippinensis and An. nivipes shared 85.2% sequence similarity and no intra-species variation was found in the nucleotide sequences of the two species. Overall, sequence data of the ITS2 marker revealed that both these species from north-east India differed by as much as have been reported from specimens of eastern Thailand.

Potential of Anopheles philippinensis-nivipes complex mosquitoes as malaria vector in north-east India.

Anopheles philippinensis-nivipes complex mosquitoes, captured in outdoor human landing catches and light traps in human dwellings from four different sites in Assam state and adjoining areas, were examined (n=1670) for the presence of circumsporozoite antigen (CSA) through enzyme linked immunosorbent assays (ELISA), using species specific capture monoclonal antibodies, of Plasmodium falciparum and two Plasmodium vivax polymorphs (Pv 210 and VK 247). In ELISA, 28 pools were found positive for CSA that accounted for minimum sporozoite rate of 1.7% (95% CI 1.11-2.41). Twenty five percent (7/28) of the positive pools were reactive for P. falciparum and between the two polymorphs of P. vivax, VK 247 was predominant with 77% (20/26) of all P. vivax positive pools. Results were suggestive of most likely involvement of Anopheles philippinensis-nivipes complex mosquitoes in malaria transmission in north-east India.

Taxonomical observations on Anopheles philippinensis/nivipes group mosquitoes in north-east India.

To examine the distribution pattern of Anopheles philippinensis and An. nivipes, mosquitoes belonging to An. philippinensis/nivipes group were collected from five sites in north-east Indian state of Assam and its adjoining areas and identified. Presence of both An. philippinensis and An. nivipes, with predominance of the latter taxon, was recorded with conformity on the basis of pupal paddle characteristics. Based on adult wing characters, 91.4% specimens were identified as An. philippinensis and 8.6% as An. nivipes with gradually increasing proportion of An. nivipes in westwardly direction. An. philippinensis/ nivipes was noted to be mid night biter, mainly exophagic and exclusively exophilic. Of the two species, An. philippinensis appears to be mostly anthropophilic and available in broken forest areas whereas An. nivipes seems to be mostly zoophilic and generally found in ecotone zones in plains areas.

Therapeutic efficacy of anti-malarials in Plasmodium falciparum malaria in an Indo-Myanmar border area of Arunachal Pradesh.

BACKGROUND & OBJECTIVES: Malaria is one of the major public health problems in the north eastern region of India. Antimalarial drug resistance is widespread and one of the important causes of recent resurgence of falciparum malaria in this region. Antimalarial drugs are seen to be used sequentially one after another in many areas of the region, when therapeutic failure is observed with a drug. In view of this, the present study was undertaken to assess the therapeutic efficacy of common antimalarial drugs viz., chloroquine, sulfadoxine+pyrimethamine (S-P) and quinine, administered sequentially to the patients with Plasmodium falciparum infection in a Myanmar bordering area of Arunachal Pradesh. METHODS: A hospital based in vivo study was carried out with 53 patients with uncomplicated falciparum malaria. All patients were first treated with chloroquine and therapeutic efficacy assessed. In case of therapeutic failure of chloroquine combination drug (S-P) was given and those showing failure with S-P combination, oral quinine was administered and followed up for 28 days to assess both clinical and parasitological responses. RESULTS: Therapeutic failure was observed with chloroquine in 83.1 per cent (44 of 53) and to both chloroquine and S-P combination drug in 44.1 per cent (19/43) patients. Further, 15.8 per cent patients (3 of 19) failed to respond even to quinine. Overall, 5.7 per cent patients (3 of 53) showed unresponsiveness to all the three drugs i.e., chloroquine, S-P combination and quinine. Asexual parasite clearance and also fever clearance was slowest with chloroquine and fastest with quinine. INTERPRETATION & CONCLUSION: The findings of this study show the presence of multi drug failure P. falciparum in Jairampur-Nampong, a western Myanmar bordering area of Arunachal Pradesh. Anti malarial drug resistance is increasing in Indo-Myanmar border areas and systematic studies need to be done to review the situation.

Malaria control in a forest camp in an oil exploration area of Upper Assam.

BACKGROUND: Assam, in north-east India, is extremely rich in hydrocarbon deposits and the oil industry is the major contributor to its economy. A large number of oil fields and related installations in Assam are located in forest areas or on their fringes where malaria is a serious problem among field staff and security personnel, adversely affecting oil production. We carried out an operational research study for one year in a forest-based industrial security camp of Dibrugarh district and developed an effective malaria control strategy for such areas. METHODS: The specific strategy was formulated and implemented after taking into account the local epidemiology of malaria, vector's ecology and malaria risk behaviour of the camp inmates. The strategy was based on reducing the man-vector contact, using deltamethrin-treated mosquito nets in conjunction with mosquito repellent cream and weekly chemoprophylaxis with 300 mg chloroquine. The impact of the strategy was monitored entomologically and epidemiologically for one year after implementation. RESULTS: The mean landing rate of Anopheles dirus, the vector mosquito in the camp area, was 5.03 per person per night during the monitoring. In spite of such a high density of the vector, the man-vector contact was effectively checked by the intervention measures adopted. As a result, the incidence of malaria in the camp was reduced by > 90% as compared to previous years and the number of malaria cases came down from 6.7 per 1000 man-nights in 1998-99 to 0.06 in 2000-01. Mortality due to malaria was completely eliminated. CONCLUSION: Control of malaria should be based on the local determinants of transmission. The use of insecticide-treated mosquito nets in conjunction with a mosquito repellent cream is a good intervention for controlling Anopheles dirus-transmitted malaria in the forests of north-east India. The control module developed on the principle of reducing man-mosquito contact is easy to implement, cost-effective and replicable in similar forest-based locations.

Physico-chemical characteristics of breeding habitats of Anopheles dirus (Diptera:Culicidae) in Assam, India.

Larval ecology of Anopheles dirus, the main vector of forest malaria in north-eastern region of India, was studied in relation to physico-chemical characteristics of its breeding habitats in a rain forest area of Assam, India. Shady stream side pools, positive for the breeding of An. dirus, had significantly higher amounts of total hardness (P < 0.024) in comparison to negative pools of similar type. When compared with An. dirus negative breeding habitats, in hot-wet as well as in cool-dry seasons, An. dirus positive shady ground pools showed higher mean values of total alkalinity, hardness and chloride content, whereas lower pH, dissolved oxygen along with higher total alkalinity and hardness were possessed by An. dirus positive stream side pools. Specificity of breeding habitats of An. dirus in relation to its ecology has been discussed.

Studies on isolation and regeneration of protoplast of thermophilic fungus Malbranchea Pulchella VAR. sulfurea

Protoplastos esféricos osmoticamente sensíveis foram isolados de micélio do fungo termofílico Malbranchea pulchella var. sulfurea pela açäo de enzima extracelular (Novozym 234) sobre a parede das células. Estäo descritas as condiçöes para obtençäo de protoplastos estáveis a partir do micélio. O uso de KCL 0,6M como estabilizador osmótico permitiu a liberaçäo do número máximo de protoplastos. A maior parte dos protoplastos foram liberados a partir de micélios cultivados por 96 - 148 h, embora micélios mais jovens liberassem protoplastos mais estáveis e maiores em tamanho. Determinou-se, também, a açäo da enzima sobre a superfície do micélio. O número máximo de protoplastos foi observado em pH 5,5. Estudaram-se também as condiçöes requeridas para a regeneraçäo dos protoplastos