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.

Establishment of two new cell lines from Bombyx mori (L.) (Lepidoptera: Bombycidae) & their susceptibility to baculoviruses.

BACKGROUND & OBJECTIVES: Lepidopteran cell cultures and baculovirus expression vector systems are becoming popular due to their potential applications in biotechnology especially for the expression of foreign proteins. Efforts were made to develop new, indigenous, cell lines from Bombyx mori larvae and pupae. METHODS: Eight to ten B. mori larvae and 10-12 pupae were surface sterilized, dissected and ovaries were removed aseptically. Ovaries were chopped finely, washed and suspended in growth medium. When the cells formed monolayers, they were subcultured and experiments were carried out. RESULTS: Two new cell lines from larval and pupal ovaries of B. mori were established in Grace's insect tissue culture medium supplemented with 20 per cent FBS (foetal bovine serum). The larval cell line consisted predominantly of epithelial-like cells (98.31%), whereas the pupal cell line had a mixed cell population of epithelial-like (71.8%) and fibroblast-like cells (27.8%). Karyology indicated a typical lepidopteran pattern in both the cell lines and had chromosome numbers ranging from 35 to 150 and 60 to 180 for larval and pupal ovaries respectively. Four-fold increase in cell number was observed in these cell lines in 7 days. Both the cell lines were found susceptible to B. mori multiple nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, but not to Helicoverpa armigera single nucleopolyhedrovirus and Spodoptera litura multiple nucleopolyhedrovirus. INTERPRETATION & CONCLUSION: These well characterized cell lines may be of immense application in biotechnology and medicine for the production of biologically active recombinant proteins to use in vaccine studies as well as in therapeutic applications.

A new cell line from the embryonic tissues of Culex tritaeniorhynchus and its susceptibility to certain flaviviruses.

A new cell line from the embryonic tissue of Culex tritaeniorhynchus mosquito was established. Morphological studies carried out at the 45th passage level (P-45) showed four different cell types viz. epithelial-like cells, fibroblast-like cells, giant cells and vacuolated cells. Karyological studies indicated diploid (2n = 6) chromosomes in majority of cells irrespective of passage level. A twelve-fold increase of cell number was observed in 10 days at P-49. The cells could be preserved in liquid nitrogen for more than 40 months. Isoenzyme profile analysis with four enzymes clearly indicated that this cell line was derived from C. tritaeniorhynchus. This cell line was susceptible to Japanese encephalitis (JEV) and West Nile viruses (WNV) but not to Dengue 1-4 (DEN 1-4) viruses. Protein of 38 K was detected in the membrane fraction of the cells from this and the C6/36 cell line, which was found to bind DEN 1-4 viruses. These data suggest that DEN viruses bind to this membrane protein and probably enter into the cells but do not continue further in the replication process.

Baculovirus studies in new, indigenous lepidopteran cell lines.

Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies.

A new cell line from the larval hemocytes of H. armigera was established in Grace's medium modified by adding lactalbumin hydrolysate and yeastolate (3.3g/l), and supplemented with fetal bovine serum (20%). The cell line was designated as NIV-HA-1195. The cell population at P-78 consisted mainly of epithelial-like cells (89.36%), fibroblast-like cells (8.31%) and giant cells (2.13%). The population doubling time was 96hr at P-8, 60hr at P-43. The chromosome number ranged from 45 to 200. The cell line is susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line.

A new continuous cell line from larval hemocytes of Spodoptera litura (F.).

A new cell line has been established from larval hemocytes of the moth, S. litura (tobacco cut worm). It took 147 days to form a monolayer and one year for the first 17 passages. At present, the culture is at 86th passage level and is designated NIV-SU-1095. Three cell types could be distinguished, viz. plasmatocytes (53%), prohemocytes (36%) and granular hemocytes (11%). The chromosome number was very high, 74% metaphase cells showed more than 100 chromosomes. The cells could be cryopreserved. The cells were susceptible to the baculoviruses, Autographa californica nuclear polyhedrosis virus and S. litura nuclear polyhedrosis virus (SLNPV). Plaques could be observed on 7th post infection day with SLNPV. Six cloned cell lines have been developed of which clone II-1F was more sensitive to both the baculoviruses compared to the original cell line.

First isolation of Japanese encephalitis virus from the mosquito, Culex tritaeniorhynchus Giles, 1901 (Diptera: Culicidae) in Gorakhpur district, Uttar Pradesh.

A strain of Japanese encephalitis (JE) virus has been isolated from a pool of female mosquitoes of C. tritaeniorhynchus, using C. bitaeniorhynchus cell line. This is the first report of JE virus isolation from mosquitoes in Gorakhpur district of Uttar Pradesh, north India.

Susceptibility of Aedes krombeini cell line to some arboviruses.

The susceptibility of the newly established Ae. krombeini cell line (NIVI-AK-453) to six arboviruses, belonging to four different families, was studied. Sindbis (SIND), Vesicular stomatitis (VSV) Chandipura (CHP) and African horse sickness (AHS) viruses multiplied in these cultures. A four-to-five-fold increase in the virus titres was observed. The maximum titre of SIND, VSV, CHP and AHS viruses were observed on 1st, 4th, 3rd and 10th post infection days, respectively. A steady and significant increase in the titre of AHS was observed over a period of ten days. The sandfly fever virus (SFV) and the tick-borne, Kaisodi virus did not multiply in the cultures.

Isolation of Japanese encephalitis virus from Culex pseudovishnui Colless, 1957 (Diptera: Culicidae) in Goa.

A strain of Japanese encephalitis virus was isolated from a pool of 54 female C. pseudovishnui Colless, 1957. The mosquitoes were collected in August 1988 during the period of epidemic of JE. This is the first report of isolation of JE virus from mosquitoes in Goa in the western coastal belt of peninsular India. In view of this isolation, C. pseudovishnui acquires greater importance, even though its density and relative prevalence during the current study was found to be far lower than C. tritaeniorhynchus.

Japanese encephalitis virus infection in mosquitoes reared from field-collected immatures and in wild-caught males.

Immature stages of mosquitoes collected in JE endemic areas of Karnataka, India between 1985 and 1987 were reared to adults and processed for the detection and isolation of Japanese encephalitis (JE) virus in an attempt to find naturally occurring vertical transmission of the virus. Males collected during 1985-1986 were also processed. A total of 15,785 adults reared from immatures and divided into 445 pools and 1,756 wild-collected males divided into 128 pools were processed using mosquito inoculation and immunofluorescence techniques. JE virus antigen was detected in 9 pools, 4 of which yielded JE virus. These were 2 pools of males and 1 pool of female Culex tritaeniorhynchus and 1 pool of male C. pseudovishnui, suggesting vertical transmission of JE virus in the mosquitoes.

Epitope analysis of strains of Japanese encephalitis virus by monoclonal antibodies.

Twenty one strains of Japanese encephalitis (JE) virus, including 16 from India, were compared antigenically on the basis of their reactivity in immunofluorescence (IF), haemagglutination inhibition (HI), ELISA with captured antigen (ECA), and neutralization (N) tests with JE monoclonal antibodies (MAbs). These MAbs represented three domains of distinct epitopes on the envelope protein, designated as Hs-1 to 4 (JE specific in HI), Hx-1 to 5 (flavivirus cross reactive in HI) and NHs-1 to 2 (non-HI JE virus specific). Fifteen of the 21 strains studied were placed in group I. These reacted with MAbs representing the three domains in all the tests indicating presence of the three types of epitopes with full functional activity. The remaining six strains were placed in group II and showed loss in HI reactivity with Hs MAbs but not with Hx MAbs. All the group II strains also reacted in IF and ECA with NHs-1. Hs epitopes in three strains, G9473 (Tamil Nadu), 641686 (Tamil Nadu) and 822199 (Karnataka), appeared to have mutated partially, indicating loss in HI reactivity with Hs MAbs only, while there was retention of other reactivities, viz., IF, ECA and to some extent N test with G9473 and 641686. The remaining three strains, 691004 (Sri Lankan), 755468 (West Bengal) and Yoken (Japan) of group II showed almost complete loss of Hs-1 and Hs-2 epitopes as there was absence of reactivity in IF, ECA and N test in addition to HI. However, Hs-3 MAb showed reactivity in IF with these strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Japanese encephalitis virus from mosquitoes collected in Karnataka state, India from 1985 to 1987.

Detection and isolation of Japanese encephalitis (JE) virus using mosquito inoculation and immunofluorescence techniques were attempted from female mosquitoes collected in JE endemic areas of Kolar and Mandya districts of Karnataka state, India, from 1985 to 1987. 65,388 mosquitoes consisting of 19 species in 1541 pools were processed. Of these, 18 pools showed the presence of JE virus antigen. JE virus was isolated from 9 pools, 3 of Culex gelidus, 2 of C. tritaeniorhynchus, and one each of C. quinquefasciatus, C. fuscocephala, C. vishnui and Anopheles peditaeniatus. Isolation of JE virus from C. gelidus, C. fuscocephala, C. quinquefasciatus and An. peditaeniatus is reported for the first time in India.