Biomedical subjects
D T Mourya
Publications and source records attributed to D T Mourya.
Vector biology of dengue & chikungunya viruses.
Explore the source record for details and available documents.
Isolation of Ganjam virus from ticks collected off domestic animals around Pune, Maharashtra, India.
Studies on viruses of zoonotic importance in certain villages around Pune were undertaken between December 2000 and January 2002. A total of 1,138 adult ticks belonging to six different species were collected off domestic animals and processed for virus isolation. Six virus isolates were obtained. All six isolates were identified as Ganjam virus by Quick Complement Fixation test and reverse transcriptase-polymerase chain reaction using RNA nucleocapsid gene amplification. Five isolates were from the pools of adult Hemaphysalis intermedia ticks, and one isolate was from a pool of adult Rhipecephalus hemaphysaloides. This is the first report of isolation of Ganjam virus from Maharashtra state of India.
Characterization of a newly established potato tuber moth (Phthorimaea operculella Zeller) cell line.
BACKGROUND & OBJECTIVE: Potato tuber moth (PTM), Phthorimaea operculella Zeller is a widely distributed, devastating pest of potatoes attacking the foliage and infest the tubers in both field and store causing serious economic damage. As application of PTM granulovirus (PTM-GV) has shown significant reduction in damage, attempts were made to develop a new cell line from this insect to grow PTM-GV for use as a biopesticide. METHODS: Approximately 100 mg of insect eggs were collected, surface sterilized and crushed gently in a boiling tube aseptically. The tissues were washed with physiological saline, suspended in growth medium and incubated stationary at 28 degrees C. Morphology of cells was studied after staining with Giemsa. Besides karyological and growth curve studies, PCR amplification was also done for rapid amplified polymorphic DNA pattern. RESULTS: A new cell line from the embryonic tissue of PTM was maintained in Mitsuhashi Maramorosch medium supplemented with 10 per cent foetal bovine serum. It is in the 78th passage level and designated as NIV-PTM-1095. Random amplified polymorphic DNA profile analysis indicated this as a new cell line from potato tuber moth and differed from the profiles of two other lepidopteran cell lines maintained in the laboratory. Three different cell types were observed at the 40th passage level and comprised of epithelial-like cells (77%), fibroblast-like cells (20%) and giant cells (3%). The chromosome number varied from 54-176. The cell line had a cell doubling time of approximately 42 h during the logarithmic phase of growth. The cell line did not support the multiplication of any of the baculoviruses used in the study. INTERPRETATION & CONCLUSION: Since the new cell line is found to replicate PTM-GV, it may be useful for the propagation of PTM-GV in large scale. Studies to scale up the production of the GV in the cell line and field trials may lead to its widespread use as an eco-friendly biopesticide.
Insect cell culture in research: Indian scenario.
Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies.
Effect of temperature and insecticide stresses on Aedes aegypti larvae and their influence on the susceptibility of mosquitoes to dengue-2 virus.
Two major factors, higher temperatures and the application of insecticides, can drastically alter the genetic structure of a vector mosquito population. Due to these two stresses, the majority of the population gets wiped out, but the ones that withstand the stress and survive are likely to pass on survivability, and have an altered physiology. Our study shows that exposures to higher temperatures and DDT during the larval stage affects their susceptibility as adult mosquitoes to the DEN-2 virus. The overall transcription and translation status of heat shock protein (Hsp70) in virus high- and low-susceptible was the same as that in other batches. In the case of a DDT-resistant (R-7) strain two bands were obtained during RT-PCRs after heat shock. These two alleles were obtained only with HY-1 in which R-7 males were used for the crosses, suggesting that the second allele is probably male sex linked. The higher expression of Hsp70 may provide DDT-resistant strains a better chance of survival high temperature environments, particularly in homozygotes and hybrids. It was also interesting to note that these strains have a significantly lower susceptibility to the virus. Wide-spread DDT-resistance and a rise in temperature above the average temperature during summer may result in a population with a low susceptibility to the virus. Several families of heat shock proteins are known to be expressed in mosquitoes, and may have a cumulative role in determining susceptibility to the virus, which itself is governed by several genes.
Effect of temperature stress on immature stages and susceptibility of Aedes aegypti mosquitoes to chikungunya virus.
A high temperature stress of 44.5 degrees C for 10 minutes on the larval stages was found to affect the susceptibility of adult Aedes aegypti mosquitoes to chikungunya virus. At this temperature, the mortality of the mosquito larvae was found to be approximately 95%, whereas a temperature greater than 45 degrees C for 10 minutes was found to be lethal. A temperature tolerant (TT) strain was developed by exposing the larvae to a temperature of 44.5 degrees C for 10 minutes at every generation for five generations. This strain was established to determine whether increase in the susceptibility was due to any selection pressure of higher temperature or to the influence of other intrinsic factors such as expression of immunoresponsive (IR) genes. Other studies on these mosquito strains showed that when maintained at 28 +/- 1 degrees C, there was no difference in the larval duration and mortality in the immature stages, but the mean survival of female mosquitoes in the TT strain was 5-6 days longer. Conversely, when mosquitoes were maintained throughout at 37 degrees C the mean survival of the mosquitoes decreased drastically in both strains, but the mean survival of females in the TT strain was 5-6 days longer compared with the unstressed controls. This increases the probability of at least one more blood meal. Fecundity of the TT strain was found to be lower than that of the control mosquitoes. Data suggest that expression of certain IR genes was affected by the heat shock. Some of these genes were up-regulated and down-regulated, which may have affected the susceptibility of mosquitoes to the virus. Although there was some selection in the temperature-tolerant individuals in the TT strain, when stressed by heat they showed expression of IR genes in a pattern similar to that in the normal controls. It appears that an increase in temperature above the average temperature of an area might help increase the proportion of virus-susceptible mosquitoes in the population. Such an increase in temperature in an endemic area would not only enhance the selection of temperature-tolerant individuals in a population having more longevity, but would also affect both intrinsic and extrinsic factors by reducing the extrinsic incubation period and increasing susceptibility of mosquitoes to viruses due to affected expression of IR genes.
The role of hemagglutinins in the midgut extracts of two lines of Aedes aegypti in their susceptibility to Dengue-2 virus.
Hemagglutinin activity (HA) was studied in the midgut extracts from highly (h) and lowly susceptible strains of Aedes aegypti mosquitoes to Dengue-2 virus (DEN-2). HA in the midgut extracts from these two isofemale strains of mosquitoes was high in as compared to (h) mosquitoes. HA was found to be higher with chicken red blood cells (RBCs) than with rabbit and human RBCs of O group. Larval midgut extracts showed higher activity than those from adult female mosquitoes. Exposure of midgut extracts to 100 degrees C for 10 mins destroyed the activity. The activity was observed between pH 6 and pH 10. HA in midgut extracts was also studied using twenty different carbohydrates; five of them showed an inhibition of HA. The inhibitory carbohydrates, when incorporated into DEN-2-infected bloodmeal, showed a reduction in the susceptibility of mosquitoes to the virus as compared to the control ones fed on the virus alone. Similarly, when these carbohydrates were incorporated in the DEN-2-infected inoculum, the inoculated mosquitoes showed a reduction in the susceptibility to the virus. HA in the virus-infected midgut extracts was higher than that in the uninfected controls. These results suggest that the presence of HA in the midgut may be one of the factors that affect the susceptibility of Ae. aegypti mosquitoes to DEN-2.
Isolation of bovine viral diarrhea virus 1, a pestivirus from autopsied lamb specimen from Tamil Nadu, India.
An epizootic of febrile illness among the Madras red breed of sheep had occurred in 1994 in Verrapuram, Chennai, India. The epizootic was suspected as Rift Valley fever (RVF)-like sickness based on clinical features. However, its etiological agent could neither be isolated nor implicated conclusively. During the post-epizootic period a male lamb died of similar clinical features and the spleen was immediately collected. Inoculation of spleen suspension in infant mouse brain yielded a virus that was serially passaged in infant mice and rhabdomyosarcoma (RD) cells. Electron microscopic observations revealed virus particles resembling flaviviruses. RT-PCR performed on extracted total RNA from infected cells and mouse brains with flavivirus-specific or RVF-specific primers gave negative results. However, an amplicon of 280 bp was obtained with pestivirus-specific primers from the 5'-UTR. Further, a nested PCR yielded a product of 157 bp. Nucleotide sequencing of the 157 bp product showed 100% homology to BVDV-1. Western blot analysis with a flavivirus envelope protein-specific MAb revealed three proteins of 33 K, 45 K and 55 K. Further studies suggested that the 33 K and 55 K proteins were glycosylated. This is the first report of isolation of BVDV-1 from a lamb in India.
Experimental transmission of Chikungunya virus by Anopheles stephensi mosquitoes.
The Ades aegypti mosquito has been considered the principal vector of Chikungunya (CHIK) virus. As CHIK epidemics usually occur in urban regions and Anopheles stephensi is another highly endophilic and anthropophilic mosquito, there is a very high probability of this mosquito to feed on CHIK virus-infected patients, to pick up and transmit the virus. Therefore the present study was conducted to test the CHIK virus transmission capability for the A. stephensi mosquito. The obtained results showed that this mosquito species is capable of transmitting CHIK virus. It is surmised that during any epidemic of febrile illness CHIK virus isolation attempts should also be made from this mosquito species.
Genotyping of Chikungunya virus isolates from India during 1963-2000 by reverse transcription-polymerase chain reaction.
Explore the source record for details and available documents.
West Nile virus: the Indian scenario.
West Nile virus (WNV) is an important arthropod borne flavivirus; usually causes a mild infection called West Nile fever (WNF) in human and horses. Mosquitoes are the principal vectors of WNV. Various Culex species are found to act as vectors in different geographical regions. The virus is maintained in a bird-mosquito cycle in nature. In India, Culex mosquitoes are tentatively incriminated as vectors of WNV. Experimental studies have shown that Culex tritaeniorhynchus, Cx. vishnui, Cx. bitaeniorhynchus and Cx. univittatus, Culex pipiens fatigans and Aedes albopictus could act as potential vectors of WNV. Transovarial transmission of WNV has been experimentally demonstrated in Culex mosquitoes. Apart from mosquitoes, the role of other arthropods is also considered in the maintenance of WNV during inter-enzootic periods. The possible role of ardeid birds in the maintenance of WNV has been described in India. Though very few clinically overt cases of human encephalitis due to WNV are observed, Japanese encephalitis virus (JEV) is found to dominate in southern India. WNF in horses has not been documented in India. JEV immunized monkeys were protected from WNV challenge and the WNV immunization was found to reduce the disease severity due to JEV. Based on the limited genome sequence analysis, the Indian isolates are grouped together under the genetic lineage-I. WNV infection is diagnosed by IgM antibody capture enzyme linked immunosorbant assay, haemagglutination inhibition test, neutralization test and reverse transcriptase-polymerase chain reaction (RT-PCR). For the effective control of Culex mosquitoes, integrated vector control strategies are recommended. Specific methods are not available for the treatment of WNV infection. However, in patients with encephalitis supportive therapy is recommended. Though a few candidate vaccines are under laboratory trial, no vaccine has been available commercially for the control of WNV infection in human and animals. In view of the global interest on WNV, this paper describes the present status of WNV in India.
A focal outbreak of Japanese Encephalitis among horses in Pune district, India.
Explore the source record for details and available documents.
Enhanced esterase activity in salivary gland and midgut of Aedes aegypti mosquito infected with dengue-2 virus.
Mosquitoes were infected by intrathoracic inoculation. About 95% head squashes were positive for dengue virus antigen on the 15th post infection day (PID). Esterase activity was determined in the homogenates prepared from the salivary glands and midguts on different PIDs of dengue virus inoculated and control mosquitoes showed that it was consistently higher in the virus-infected batches.
A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae).
A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.
Persistence of dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes.
Progeny of Aedes aegypti mosquitoes infected intrathoracically with dengue-3 virus was reared to subsequent generations. In each generation, blood-fed females were confined individually and the eggs obtained from the transovarially infected females were pooled. The seventh generation obtained from the infected parental mosquitoes showed that virus could persist in mosquitoes in successive generations through transovarial passage. The rate of vertical transmission initially increased in the few generations (F1-F2), but in subsequent generations it was found to be steady. Parental mosquitoes inoculated with virus showed higher mortality than the diluent-inoculated controls. There was an increase in the larval duration of transovarially infected batches at the seventh generation when compared with uninfected control mosquitoes. The fecundity and fertility of the transovarially infected batches of mosquitoes was also affected when compared with the controls. This is the first report demonstrating persistence of dengue virus in the successive generations of mosquitoes infected through vertical transmission. These observations, which have great epidemiologic importance, suggest that vector mosquitoes may play an important role in the maintenance of virus in nature, and that mosquitoes may act as reservoirs of these viruses.
Effect of anticoagulants on the susceptibility of Aedes aegypti mosquitoes to dengue virus infection.
Explore the source record for details and available documents.
Development of monoclonal antibody based antigen capture ELISA to detect chikungunya virus antigen in mosquitoes.
BACKGROUND & OBJECTIVES: Chikungunya (CHIK) virus has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic which occurred in 1971. For active surveillance, it is necessary to have a test, which can detect the virus from a large number of field-collected mosquitoes. METHODS: The present study describes the standardization of monoclonal antibody (MAb) based antigen capture ELISA to detect chikungunya virus antigen from the mosquitoes. CHIK virus antigen from suspension of experimentally infected mosquitoes and their progeny was captured on mouse polyclonal antibody, while biotinylated CHIK Mab was used as a probing antibody. CHIK virus antigen in the head squashes of virus inoculated mosquitoes was detected using indirect immunofluorescence antibody (IFA) test for confirmation of ELISA results. RESULTS: The ELISA test was sensitive enough to detect antigen even if a small fraction of a single infected mosquito homogenate was incorporated in the test. The IFA test failed to detect CHIK antigen in 10 and 25 microliters of suspension whereas with ELISA it was detected in all the samples. Progeny of Aedes aegypti and Ae. albopictus mosquitoes infected with chikungunya virus did not show the possibility of existence of transovarial transmission. INTERPRETATION & CONCLUSION: This test is rapid and simple since it can be completed in two days as compared to the conventional mosquito inoculation and IFA techniques, which require at least 10 days. There is an additional advantage with this test that a large number of samples can be processed, and the remaining homogenate of the mosquitoes can be used for screening other viruses. Experimental data raised using this test showed that transovarial transmission of this virus does not occur in these vector species.