|Year : 2016 | Volume
| Issue : 4 | Page : 258-265
An observation on direct changes in Aedes albopictus midgut cells by Rhus tox 6C in relation to dengue virus infection
Moonmoon Sinha1, Enakshi Roy1, Satadal Das1, Debabrata B Sarkar1, Debadatta Nayak2, Anil Khurana2, Raj Kumar Manchanda2
1 Dr. Anjali Chatterjee Regional Research Institute for Homoeopathy, Kolkata, West Bengal, India
2 Central Council for Research in Homoeopathy, New Delhi, India
|Date of Web Publication||17-Nov-2016|
Dr. Anjali Chatterjee Regional Research Institute for Homoeopathy, 50, Rajendra Chatterjee Road, Kolkata - 700 035, West Bengal
Source of Support: None, Conflict of Interest: None
Background and Objectives: In mosquito vectors, dengue virus (DENV) invasion occurs through midgut cells, but available mosquito cell lines for in vitro study of DENV are prepared from eggs or larvae, which are not appropriate models, to study its infectivity. Hence, we developed a new primary cell culture, from Aedes albopictus mosquito midgut, and standardized it for in vitro study of DENV, with an aim to find out any possible role of homoeopathic medicines, in preventing or reducing DENV invasiveness in these midgut cells. This midgut primary cell culture demonstrated prominent cytopathic effects on infection with wild DENV isolated from dengue-infected patients in viremic phase. Materials and Methods: In this paper, we observed the direct effect of homoeopathic medicine Rhus toxicodendron 6C (Rhus tox 6C) (ultra dilution of 10−12 ) on this primary cell culture, to find out significant changes, to be used as baseline data in future experiments to observe possible role of Rhus tox 6C against DENV infection in these cells. Hence, these direct changes may be a prerequisite for the action of this medicine against DENV invasion; as this is one of common repertoire homoeopathic medicines used against dengue fever. Conclusion and Discussion: In our experiments, we found that Rhus tox 6C could increase cell size and help organization of cells on the solid surface as observed under scanning electron microscope although the total number of cells was decreased. Moreover, Rhus tox 6C treated cells were healthier as indicated by less number of deformed, clump, and diploform cells.
Keywords: Aedes albopictus midgut cell line, Arbovirus, Rhus tox 6C
|How to cite this article:|
Sinha M, Roy E, Das S, Sarkar DB, Nayak D, Khurana A, Manchanda RK. An observation on direct changes in Aedes albopictus midgut cells by Rhus tox 6C in relation to dengue virus infection. Indian J Res Homoeopathy 2016;10:258-65
|How to cite this URL:|
Sinha M, Roy E, Das S, Sarkar DB, Nayak D, Khurana A, Manchanda RK. An observation on direct changes in Aedes albopictus midgut cells by Rhus tox 6C in relation to dengue virus infection. Indian J Res Homoeopathy [serial online] 2016 [cited 2017 Feb 25];10:258-65. Available from: http://www.ijrh.org/text.asp?2016/10/4/258/194322
| Introduction|| |
Among the several life-threatening arboviral diseases, of the tropical and subtropical countries, the most common and fatal disease is dengue fever caused by an 11 kDa, positive-stranded RNA virus called dengue virus (DENV).  This Flaviviridae virus infection is the cause of about 20,000 annual deaths  and about 500,000 cases of severe dengue infections - dengue haemorrhagic fever/dengue shock syndrome. Over the past 50 years, the incidence of DENV infection has increased to 30-fold, and according to the WHO, about 50100 million people are infected with DENV each year, in over 100 countries worldwide.  The four genotypes (DENV 14) of this virus are transmitted by mosquitoes of Aedes sp., mainly Aedes aegypti and Aedes albopictus. The main clinical presentations of dengue fever are pyrexia, joint pain, skin rashes, headache, muscle pain, and anorexia. 
Although being a severe threat to human life, the treatment modality of dengue fever is a general one; without any specific antiviral agent, to cure the disease, and in spite of continuous trials on dengue fever vaccines, no official vaccine program has been started in any of the affected countries so far. Furthermore, except recently developed AG 129 mice (129 Sv mice deficient in alpha, beta, and gamma interferon receptors), absence of easily available proper animal model makes it more difficult, to study the pathogenesis of DENV  Homoeopathic medicines such as Rhus tox; Eupatorium perfoliatum; Belladonna; Arsenicum album; Bryonia and many others in different potencies are being used to treat DENV infected people, in different parts of India and other countries such as Brazil, Central America, Thailand, Sri Lanka, Pakistan and Cuba , Thus, it appears essential to study the detailed effects of these medicines at molecular levels, on in vitro cell lines and in animal models in vivo. Hence, we have developed a protocol, which will be followed in several stages, that involves preparation of primary cell culture from midgut of A. albopictus, followed by application of homoeopathic medicines on these cells, and subsequent studies of the effects of homoeopathic medicines on these cells, after challenging with DENV. In this paper presents preliminary results of the first part of this protocol, where we observed a direct effect of a homoeopathic medicine Rhus tox 6C (prepared from Toxicodendron radicans) [Figure 1] and [Table 1] on a newly prepared primary midgut cell culture of A. albopictus.
After a comprehensive review of literature, we noticed that propagation of DENV is best observed in mosquito midgut cells, which was originally observed by researchers in organ culture, as it is the primary target site of viral invasion.  After entry of DENV in mosquitoes, main pathological changes are found in the midgut and salivary gland cells. ,,, Among them, it was presumed that study of midgut cells is the best tool which mimics the real biological interaction between host, vector, and pathogen. 
Available mosquito cell lines are either prepared from larvae or eggs,  and these established cell lines are commonly used for propagation of DENV  and related flaviviruses although these cells do not represent the natural host-pathogen interaction, which could be observed in the midgut cells of mosquitoes.
Hence, with an aim to study the real biological interaction of DENV with the vector as well as the host cells, a new attempt has been made in this initial phase of our project, to expand cell lines from midgut of locally available wild A. albopictus mosquitoes in West Bengal. This new primary cell culture was standardized by us, and it is expected to facilitate studies involving interactions of viral antigens with specific receptors, microRNA, silencing genes, and other molecular markers; a standardized midgut cell culture will also assist in comparative studies of pathogenicity, as well as drug development toward many viral and parasitic diseases, in which mosquitoes are the only vectors. The standardized protocol of the preparation of A. albopictus midgut cell culture developed by us and the direct effect of a homoeopathic medicine Rhus tox 6C on these cells are presented here.
| Materials and Methods|| |
Collection and Sterilization of Aedes albopictus Mosquitoes
Mosquitoes were trapped from the surrounding areas of DACRRIs building, near Dakshineswar (22.6554° N, 88.3579° E) by mosquito nets. The collected mosquitoes were identified by Professor A.K. Hati, Ex-Director and Head, Department of Entomology, School of Tropical Medicine, Kolkata, based on classical descriptions.  The collected mosquitoes were anesthetized  by keeping them at low temperature (~4°C). The mosquitoes were washed with sodium dodecyl sulfate solution (10% [v/v]), followed by distilled water, sodium hypochlorite solution (0.1% [v/v]), and again distilled water for 2 min in each solution. 
Mosquito Dissection and Separation of Mosquito Midgut
The mosquito was then placed on a drop of phosphate buffer saline (PBS) on glass slide, followed by dissection and separation of the midgut. The separated midgut was then sterilized by immersing consecutively in insect physiological solution (IPS) with sodium hypochlorite solution, IPS without sodium hypochlorite solution, and distilled water as illustrated in [Figure 2]. After sterilizing the midgut, it was gently macerated to make homogeneous cell suspension in Dulbecco's Modified Eagle's Medium (DMEM) culture medium. 
Morphological Study of Midgut
The midgut was treated with 1 M NaOH solution, and after 30 min, the midgut was observed microscopically at ×100 magnification.
Histological Study of Midgut of Aedes albopictus Mosquitoes
Paraffin blocks of the midgut were prepared, sections were made in a microtome, and then the sections were stained with hematoxylin and eosin (H and E) staining method and mounted with a mixture of distyrene, plasticizer and xylene.
Aedes albopictus Midgut Cell Culture
After mosquito dissection and separation of midgut, the isolated midgut cells were inoculated in 6-well culture plates (1.2 × 10 6 cells/well) containing DMEM culture medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), sodium bicarbonate (3.7 g/L) (Sigma, USA), penicillin-streptomycin (100 U/mL and 100 μg/mL) (Gibco), and antimycotic solution (amphotericin B-0.25 μg/mL) (Gibco). The culture plates were kept in incubation at 28°C till the cells became confluent (~72 h).
Subculturing of Aedes albopictus Midgut Cells
At around 75% confluency, the cells were counted in a hemocytometer chamber. Fresh DMEM culture medium (Gibco, USA) supplemented with 10% FBS (Gibco), sodium bicarbonate (3.7 g/L) (Sigma, USA), penicillin-streptomycin (100 U/mL and 100 μg/mL) (Gibco), and antimycotic solution (amphotericin B-0.25 μg/mL) (Gibco) was added to the wells. Then, the cells were aspirated gently and removed from the culture flask and subcultured in 1:2 ratio in DMEM culture medium supplemented with 10% FBS, sodium bicarbonate, penicillin-streptomycin and antimycotic solution and maintained at 28°C and 5% CO 2 for 72 h to reach confluency.
Preliminary Observation of Cytopathic Effect of Wild Dengue Virus on Aedes albopictus Primary Midgut Cell Culture
Serum samples collected aseptically from three patients suffering from dengue fever for 2-3 days with NS1 Ag levels more than 30 units, indicating high levels of viremia, were used to infect confluent A. albopictus midgut cell culture. The infected cell culture was incubated at 28°C and 5% CO 2 for 2-7 days or until cytopathic effect (CPE) appeared. Control studies were also made with three NS1 Ag negative serum samples.
Scanning Electron Microscopy Analysis of Cells of Midgut of Aedes albopictus
For electron microscopy, the sample was washed with PBS followed by fixation in glutaric dialdehyde and subsequently with osmic acid on glass slides of definite measurements. The cells were then subjected to increasing gradient of alcohol followed by incubation in a CO 2 chamber to dehydrate the sample. Finally, the glass slides containing sample were mounted on studs, and the samples were coated with gold in S150 sputter coater of Edwards. After gold coating, the samples were observed and analyzed in FEI QUANTA 200 (Bose Institute, Rajabazar, Kolkata, West Bengal, India) at high vacuum mode. The cells on the "test" (Rhus tox 6C treated) and the "control" (succussed alcohol treated 6C) slides were observed under scanning electron microscopy (SEM).
Cell viability assay by trypan blue exclusion method
Midgut cell cultures (1.5 × 10 5 cells/mL) were prepared in 96-well tissue culture plates containing DMEM culture medium and incubated at 28°C and 5% CO 2 . The cell viability was determined before inoculation by mixing equal volume of 0.4% trypan blue solution (Sigma, USA) (1:1) to cell suspension. Viable and dead cells were counted under phase contrast microscope. The percentage of cell viability was calculated using the following formula:
% viability = (Live cell count/total cell count) × 100
Cell viability assay by cell counting kit 8 method
Midgut cell cultures were prepared, and cells were inoculated in serial dilutions (1.6 × 10 3 cells, 8 × 10 2 , 4 × 10 2 , 2 × 10 2 , 1 × 10 2 ) in 96-well plates containing DMEM culture medium and incubated for 0, 12, and 48 h. After incubation, 10 μL of water soluble tetrazolium solution of cell counting kit 8 was added to each well and incubated for 3 h at 28°C and 5% CO 2 . After incubation, the absorbance of each well was measured at 450 nm and was represented graphically against a number of cells.
Application of Rhus tox 6C on Aedes albopictus Primary Midgut Cell Culture
In this experiment, 100 μL of Rhus tox 6C was applied to the 6 well "test" cell culture plates and 100 μL of succussed alcohol 6C to 6 well "control" cell culture plates. Then, the cell culture plates were observed under inverted microscope at ×400 magnification.
Then, to study the morphological characteristics of the cells, the cells of both "test" and "control" groups were stained with Leishman's stain and the morphological changes were evaluated statistically.
Under inverted microscope, different microscopic fields were selected at random. At ×400 magnification, the morphological characteristics of the cells treated with Rhus tox 6C ("test") were studied along with cells treated with succussed alcohol 6C ("control"). The parameters that were taken into consideration were total number of cells, number of round cells, number of deformed cells, number of diploform cells, number of chains, and number of clumps (≥3 cells). Based on the above parameters, statistical analysis of the effect of Rhus tox 6C on the cells of the "test" cell culture plates was done compared to the cells of the "control" cell culture plates.
| Results|| |
The morphological characteristics of A. albopictus midgut tissue when studied under inverted microscope at ×100 magnification after treatment with 1 M NaOH solution showed a network of branching tubules known as tracheoles on the surface of midgut [Figure 3]a and distinct arrangement of residential cells containing rectangular-shaped cells, which were identified as columnar epithelial cells, a small number of goblet cells, regenerative cells, and round stem cells [Figure 3]b. Histopathological analysis after H and E staining showed pseudostratified lining of cells, which was sometimes discontinuous. The smooth muscle cells and fibrous cells were also seen organized representing muscular and serous coats. The predominant mucous columnar cells were found in clumps.
|Figure 3: (a) Tracheoles were commonly found on the surface of midgut as branching tubules (×400). (b) Pseudostratified cellular morphology of different cells on midgut surface of Aedes albopictus|
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After application of NS1 Ag positive serum containing wild DENV, typical DENV-induced CPE [Figure 4]b was observed within 48 h which was remarkably absent in all control experiments with NS1 Ag negative serum [Figure 4]a. The main changes were markedly swollen cells in big clumps [Figure 4]b.
The electron microscopic study of primary culture of A. albopictusmidgut cells showed that the cells of "test plate" (Rhus tox 6C treated cells) [Figure 5]b were large in size, more in number, and were more organized than the cells of "control plate" (succussed alcohol 6C treated cells) [Figure 5]a.
The cell viability was determined by trypan blue assay and the % viability was found to be 80% [Table 2] at the time of cell inoculation in cell culture plates.
The rate of proliferation of primary midgut cells in culture over increasing incubation time is shown in [Figure 6]. The viability assay indicates a higher growth rate at 48 h in comparison to growth rate at 0 and 12 h.
|Figure 4: (a) Cytopathic effect of primary midgut cells after 48 h posttreatment with NS1 Ag negative serum. (b) Cytopathic effect of primary midgut cells after 48 h postinfection with wild dengue virus (NS1 Ag positive serum)|
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|Figure 5: (a) Cells of "control" (succussed alcohol treated 6C) plate under scanning electron microscopy (×6000). (b) Cells of "test" (Rhus tox 6C treated) plate under scanning electron microscopy (×5000)|
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After observations of all the four sets in this experiment, i.e., on observation of cells of the "test" cell culture well, compared to the "control" cell culture well under inverted microscope at ×400 magnification in all the sets, it was identified that the cells treated with Rhus tox 6C [Table 3] and [Figure 7] were mostly single cells and few were in clumps compared to the cells of the "control" cell culture well, where most cells were in clumps. In addition to that, the structure of the cells of the "test" cell culture well was found almost intact and healthy, in contrast to the mostly deformed cells of the "control" cell culture well [Figure 8].
|Figure 6: Viability assay of primary midgut cell culture with increasing time of incubation|
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|Figure 7: Statistical interpretation of Rhus tox 6C induced changes in cell culture derived from midgut of Aedes albopictus|
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|Figure 8: (a) Microscopic view (×400) of midgut cells of control plate (succussed alcohol 6C) showing many deformed and clumped cells. (b) Microscopic view (×400) of midgut cells of test plate (Rhus tox 6C treated) showing mostly normal cells|
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|Table 3: Statistical interpretation of Rhus toxicodendron 6C induced changes in cell culture derived from midgut of Aedes albopictus|
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| Discussion|| |
Arboviruses, gametocytes of malaria parasites, and microfilaria enter the mosquito through an infectious blood meal, the pathogens then cross the peritrophic membrane, pass through the membranous microvilli-associated network, make contact with microvillar surface, penetrate into the midgut pseudostratified epithelium, and finally disseminate and multiply in other parts of the mosquito. According to the abundance of microvilli, the cells of midgut are divided into two types - the predominant type is the columnar cells with dense microvilli, which are interspersed with the other variety of cells with fewer microvilli, found particularly in the posterior part of the midgut, where invasion of pathogens commonly occurs. These cells in the posterior part of the midgut are with plenty vesicular ATPase. The pseudostratified lining cells of the midgut are placed on the basement membrane, surrounded by muscle fibers, nerve fibers, and tracheoles. According to predominant function, the midgut cells are classified into secretory cells containing basal membrane labyrinth, common absorptive cells without basal membrane labyrinth, and smaller endocrine cells which are again divided into various types on the basis of the presence of secretory granules, electron density, and reactivity to specific antipeptide hormone antisera. Besides these cells, there are also goblet cells and small, basally located regenerative cells originating from stem cells. The arrangement of cells is illustrated in [Figure 3]. A semi-stable (usually all primary cultures are semi-stable as within months dividing cell population gradually decreases) balance between stem cell proliferation, differentiation to columnar and goblet cells, and death of these mature cells occurs in a primary midgut cell culture as observed by us.
A crucial point of microbial transmission is their attachments on receptors of midgut cells which are oligosaccharides on midgut glycoproteins. ,, A high proportion of N-linked GlcNAc- and GalNAc-terminal oligosaccharides are present in these glycoproteins.  Sugar epitopes may be used as targets to block transmission of infectious agents. A broad understanding of these sugar structures is essential for experiments based on these targets. Mosquito cell lines derived from larvae have already been used to study the lectin-mediated adhesion system.  In midgut of hemipteran insects, an unusual extracellular lipoprotein membrane is found covering the microvilli of midgut cells. This membrane is known as perimicrovillar membrane containing α-glucosidase, and the space between the perimicrovillar membrane and the microvillar membranes is known as the perimicrovillar space;  however, this space is not reported in mosquitoes.
Thus, a new mosquito midgut cell line developed by us may be an ideal in vitro model for the study of biological characteristics of DENV propagation in a natural way, and future study of possible interactions of different homoeopathic medicines at all pivotal molecular steps inside the cell.
Primary midgut cell cultures were semi-stable for about 1 month, with a slowly decreasing population of dividing cells. During this 30 days period, about 10% of existing columnar and goblet cells are dying and are replaced by differentiating round stem cells.
| Conclusion|| |
In this study, we observed that Rhus tox 6C increases cell growth and organization in the culture plate as observed under SEM, while the rate of deformity and clumping of cells have also decreased.
There is no study on direct action of Rhus tox 6C on any cell line till date, and only a few studies have been done on its biological activities. Thus, this study will open up a new avenue of future studies with this new primary cell culture, for the benefit of the humanity at large.
We acknowledge the advice and guidance received from Dr. Rathin Chakraborty, Member, Scientific Advisory Committee, CCRH, Project Officer, DACRRI, Kolkata, and Mayor, Howrah Municipal Corporation. This paper is based on the 1 st year's work of CCRH project entitled "Effect of homoeopathic preparations on dengue virus infection on mosquito cell line and in suckling mice."
Financial Support and Sponsorship
The total financial and material support was provided by Central Council for Research in Homoeopathy (CCRH), New Delhi - 58.
Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]