Dengue and dengue haemorrhagic fever: Indian perspective.

The relationship of this country with dengue has been long and intense. The ?rst recorded epidemic of clinically dengue-like illness occurred at Madras in 1780 and the dengue virus was isolated for the ?rst time almost simultaneously in Japan and Calcutta in 1943-1944. After the ?rst virologically proved epidemic of dengue fever along the East Coast of India in 1963-1964, it spread to allover the country.The ?rst full-blown epidemic of the severe form of the illness,the dengue haemorrhagic fever/dengue shock syndrome occurred in North India in 1996. Aedes aegypti is the vector for transmission of the disease. Vaccines or antiviral drugs are not available for dengue viruses; the only effective way to prevent epidemic degure fever/dengue haemorrhagic fever (DF/DHF) is to control the mosquito vector, Aedes aegypti and prevent its bite. This country has few virus laboratories and some of them have done excellent work in the area of molecular epidemiology,immunopathology and vaccine development. Selected work done in this country on the problems of dengue is presented here.

Effect of pretreatment with chromium picolinate on haematological parameters during dengue virus infection in mice.

BACKGROUND & OBJECTIVE: Dengue virus (DV) has caused severe epidemics of dengue fever (DF) and dengue haemorrhagic fever (DHF) and is endemic all over India. We have earlier reported that exposure of mice to hexavalent chromium [Cr(VI)] compounds increased the severity of dengue virus infection. Trivalent chromium picolinate (CrP) is used worldwide as micronutrient and nutritional supplement. The present study was therefore, carried out to investigate the effects of CrP on various haematological parameters during DV infection of mice. METHODS: The Swiss Albino smice were inoculated with dengue virus (1000 LD50, intracerebrally) and fed with chromium picolinate (CrP) in drinking water (100 and 250 mg/l) for 24 wk. Peripheral blood leucocytes and other haematological parameters, and spleens were studied on days 4 and 8 after virus inoculations and the findings were compared with those given only CrP and the normal control age matched mice. RESULTS: CrP in drinking water for 24 wk had no significant effects on peripheral blood cells of mice. On the other hand, there was significant decrease in different haematological parameters following inoculation of normal mice with DV. In CrP fed mice the effects of DV infection were abolished on most of the haematological parameters. INTERPRETATION & CONCLUSION: The findings of present study showed that the adverse effects of DV infection, specially on platelets and leucocytes, were abrogated by pretreatment of mice with CrP. The therapeutic utility of CrP in viral infections including dengue needs to be studied in depth.

Macrophage and dengue virus: friend or foe?

The cells of monocyte-macrophage (Mphi) lineage play important roles both in innate and adaptive immune responses. They are the first line of defence in body and their job is to phagocytose a foreign invader, the pathogen, digest it and remove it. Mphi help body in mounting the antigenspecific immune response by presenting the digested pathogen antigen in conjunction with major histocompatibility complex (MHC) class II molecules to recruit B and T lymphocytes response. Usually Mphi succeed in their job of eliminating most pathogens from the body but sometimes the pathogen strikes a "friendship" with them and starts using them for its benefit. A number of pathogens, including dengue virus (DV), subvert Mphi and use them for their replication, increasing the severity of damage to the body. This duality may be related to the fact that Mphi serve as efficient host cell for DV replication, in addition to being responsible for innate immunity and for initiating adaptive immune responses. This review gives a brief overview of the various roles of Mphi (enmity and friendship) during dengue virus infection.

Effects of dengue virus infection on peripheral blood cells of mice exposed to hexavalent chromium with drinking water.

BACKGROUND & OBJECTIVE: The occupational and non-occupational exposure to hexavalent chromium Cr (VI) is common. The effect of chromium compromises the immune response of the host. Dengue virus (DV) infection causes various changes in the peripheral blood cells. It is, therefore, possible that the chromium toxicity may affect the disease process during DV infection. The present study aims to study the effects of dengue virus infection on peripheral blood cells of mice fed Cr (VI) with drinking water. METHODS: One group of mice was given ad libitum drinking water containing Cr (VI) and the other group used as the normal control mice was given plain water to drink. At the 3, 6 and 9 wk of Cr (VI) drinking, a set of mice from each group was inoculated intracerebrally (ic) with DV and studied at the 4th and 8th day post inoculation. RESULTS: It was observed that Cr (VI) drinking led to reduction in lymphocytes, haemoglobin and the haematocrit values while the granulocyte, monocyte and platelet counts were increased. On the other hand, most of the parameters were decreased following inoculation of normal mice with DV. In Cr (VI)-fed mice the effects of DV infection were minimal. The most significant finding of these experiments was that the reduction in platelet counts following inoculation with DV was markedly less in Cr (VI)-fed mice than that in DV-inoculated normal control mice. INTERPRETATION & CONCLUSION: Cr(VI) compounds have been declared as a potent occupational carcinogen. On the contrary, Cr(III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been shown to exhibit health benefits in animals and humans. Whether therapeutic doses of chromium (III) compounds may be able to prevent the DV-induced fall in platelet counts, needs to be investigated.

Dengue vaccines: problems and prospects.

The extent of cumulative disease burden caused by dengue virus has attained an unprecedented level in recent times with sharp increase in the size of human population at risk. Dengue disease presents highly complex medical, economic and ecologic problems. The surge in publications on the development of dengue vaccines, taking advantage of new generation of biotechnology techniques indicates the profound interest and urgency in the scientific and medical communities in combating this disease. This review summarizes the importance of critical subjects like pathogenesis of dengue haemorrhagic fever and inadequacy of animal model that have adversely affected dengue vaccine development. Further, the remarkable progresses so far made in dengue vaccine research not only employing a diverse range of new strategies but also re-using old techniques to improve the existing vaccines, have been presented. The efficacy and safety of some of the new vaccine candidates have been evaluated and proven in human preclinical/clinical trials. Besides the technical advancement in vaccine development, vaccine safety and vaccine formulation have been examined.

Gut microflora & toxic metals: chromium as a model.

The gastrointestinal tract (GIT) is exposed to various environmental pollutants including metals, that contaminate food and water which may have toxic effects on body. GIT has large amount of microbes that live in symbiosis and help the host in different ways. The resident gut microflora have a significant role to play in detoxification and elimination of the harmful metals from the body. Chromium is a naturally occurring heavy metal found commonly in environment in trivalent (Cr III) and hexavalent (Cr VI) forms. Cr (VI) compounds have been shown to be potent occupational carcinogens. The reduction of Cr (VI) to Cr (III) results in the formation of reactive intermediates that together with oxidative stress and oxidative tissue damage, and a cascade of cellular events including modulation of apoptosis regulatory gene p53 contribute to the cytotoxicity, genotoxicity and carcinogenicity of Cr(VI)-containing compounds. The data discussed here with reference to chromium show that gut microflora have a marked capacity to cope with the increased load of ingested metals and may contribute significantly in the protection against metal toxicity.

A clinical study of the patients with dengue hemorrhagic fever during the epidemic of 1996 at Lucknow, India.

This paper describes the clinical findings in 206 patients with dengue fever (DF) or with dengue hemorrhagic fever (DHF) during the epidemic of 1996 at Lucknow. The age group affected most was 11 to 30 years and 21% of the patients were less than 10 years old. The male:female ratio was 1.9:1. The onset was abrupt in all the patients, severe frontal headache was observed in 97%, myalgia in 90%, skin rash in 40%, vomiting in 29% and arthralgia in knee and hip joints in 9%. Anuria was seen in two patients. Lymphadenopathy was noted in 14%, hepatomegaly in 4%, being associated with mild jaundice in one patient, and splenomegaly in 2% of the patients. Involvement of the heart and lungs was seen in one patient each and no case with encephalitis was recorded. Hemorrhages from various sites were observed in 54% patients and 17 patients had profound shock. The commonest bleeding site was gums. Profound shock was preceded by various warning signs, the commonest being sudden hypotension. Among the patients with profound shock the mortality was 47% while the overall fatality rate was 3.8%. A number of the risk factors existed for a long time in this part of the world, but what precipitated the present epidemic at this time, is not known.

Internalization of dengue virus-induced suppressor cytokine during transmission of the suppressor signal via macrophage.

In dengue type 2 virus (DV)-induced suppressor T cell cascade TS1 cells secrete a suppressor cytokine (SF) which acts via syngeneic macrophages (M phi) to recruit TS2 cells. SF binds to both high and low affinity receptors (SF-R) on M phi. In the present study the fate of SF in M phi during transmission of suppressor signal is investigated. It was observed that SF bound to high affinity receptors internalized through receptor mediated endocytosis. This was inhibited by pretreatment of M phi with anti-SF-R-antiserum and didansylcadaverine, a potent inhibitor of endocytosis. Internalized SF was degraded by lysosomal activity as shown by inhibition of suppressor activity by pretreatment of M phi with monensin and NH4Cl. Degraded SF was transported to a site other than SF-R on M phi membrane for recruitment of TS2 cells. This was inhibited by anti-SF-antiserum. Transmission of suppressor signal is inhibited if M phi are treated first with H-2K-mAb and then with SF (shown earlier) but when M phi were treated first with SF and after 1 hr with H-2K-monoclonal antibody, the inhibition did not occur. As SF requires binding to H-2K and SF-R for mediation of suppression, the binding of H-2K occurred with degraded SF within the cell. Thus SF is internalized, degraded and binds to H-2K antigen before its recognition by native T cells.

Role of nitric oxide in transmission of dengue virus specific suppressor signal.

Production of NO2- was maximum when peritoneal M phi was incubated with SF (40 mg) for 45 min. Pretreatment of M phi with anti-SF-antisera inhibited production of NO2- Pretreatment of M phi with NG monomethyl L-arginine (L-NMA) or arginase, an inhibitor of L-arginine dependent pathway, inhibited production of NO2- and transmission of suppressor signal in a dose-dependent manner. This indicates that NO and Ca2+ serve as intracellular signal in transmission of DV-induced suppressor signal.

ELISA for detection of dengue virus-induced cytokine and its antibody.

ELISA technique has been standardized for detection of a dengue type 2 virus (DV)-induced cytokine, the cytotoxic factor (CF) and CF-specific antibodies. The performed ELISA was found to be sensitive (90.9%), specific (92.5%), accurate (91%) and reproducible and was able to detect a minimum of 7 ng/ml CF in the test samples. The technique was used to detect CF in DV inoculated mouse sera and DV-infected mouse spleen cell culture fluids. Significant utility of the test was detection of a CF-like cytokine in the sera of human cases of dengue haemorrhagic fever.

Identification and characterization of receptor for dengue virus-induced macrophage cytotoxin.

The present study was undertaken to identify the receptor for dengue virus type 2 (DV) induced macrophage cytotoxin (CF2) on mouse peritoneal macrophages (MPhi). The binding of 125I-labelled CF2 to MPhi was saturable (15 nM), reversible, temperature, pH- and time-dependent. The saturation concentration was similar to that causing cell death. Scatchard analysis showed the presence of intermediate type of affinity receptor and the number of receptor sites was 1.1 x 10(6) per cell with dissociation constant of 14.28 nM.

HIV-2 prevalence in Uttar Pradesh.

Serum samples collected since 1989 with various patterns of reactivity for human immunodeficiency virus (HIV)-1, on the basis of screening ELISA and confirmatory Western blot (WB) test, were subjected to the detection of HIV-2 infection based on screening dot immunoassay and confirmatory WB for HIV-2. Significant prevalence of HIV-2 infection was (37.03%) among sera reactive for HIV-1 by ELISA but indeterminate by Western blot, compared with sera reactive for HIV-1 by ELISA and WB (3.29%) or negative by WB (2.63%). Out of 16 HIV-2 positive sera, 5 (31.25%) showed evidence of concomitant HIV-1 infection. This study demonstrates evidence of HIV-2 infection as early as 1989, earlier than reported so far from India.

Role of calcium in induction of dengue virus-specific helper T cells.

Dengue type 2 virus (DV) induces the generation of T helper cells (TH) in the mouse spleen. These TH on adoptive transfer to DV primed syngeneic mice enhance the clonal expansion of antigen-specific antibody plaque forming cells. The present study was undertaken to investigate the role of M phi and Ca2+ in the induction of DV-specific TH. The results show that M phi are obligatory for the induction of DV-specific TH. It was observed that DV antigen stimulate the influx of calcium ion (Ca2+) into T cells as shown by radiolabelled (45Ca) and the influx of Ca2+ increases with time of incubation, reaching a maximum level after 1 hr of incubation. The proliferation of T cells was found to be dependent on presence of Ca2+ as it was inhibited by Ca channel blocking drugs and in the absence of Ca in the medium. Thus showing that Ca2+ plays an important role in the induction of DV-specific TH cells.

Flow cytometric analysis of T4:T8 splenic cell during dengue virus infection of mice.

The present study was undertaken to investigate the effect of dengue type 2 virus (DV) and DV-induced cytokines (CF and CF2) on T lymphocyte subpopulations of spleen by flow cytometry. Following DV-ic inoculation in mice the percent number of CD4+ and CD8+ lymphocytes in the spleen was reduced, the peak reduction in both was observed on the 6th day post-inoculation (p.i.). Intravenous inoculation of CF or CF2 in mice also decreased the percent number of CD4+ as well as CD8+ T lymphocytes subpopulation in the spleen, the maximum reduction being observed at 1 and 2 hr, respectively. The reduction in T lymphocyte subpopulation by CF and CF2 was found to be dose dependent. Thus, the alterations of T lymphocyte subpopulations during DV infection are mediated via cytokines.

Transmission of dengue virus-specific suppressor signal depends on the presence of calcium.

The present study was undertaken to investigate the role of calcium in the production of dengue type 2 virus (DV) induced suppressor cytokine (SF) and in transmission of the suppressor signal via syngeneic macrophage (M phi) to recruit the second subpopulation of suppressor T(TS2) cell. Effect of calcium channel blocking drugs, Verapamil and Nifedipine, on the production and activity of SF was investigated. The production of SF was inhibited in a dose dependent manner by the calcium channel blockers. The suppressor activity of SF was also inhibited by calcium channel blocking drugs. SF could not be produced when spleen cells were cultured in a calcium free medium, the production being restored on substitution with calcium chloride. Treatment of M phi with the calcium channel blockers inhibited the transmission of the suppressor signal from TS1 to TS2 cells in a dose dependent manner. The influx of calcium during transmission of the suppressor signal was studied by measuring the 45Ca uptake. Calcium channel blocking drugs inhibited the 45Ca uptake by T lymphocytes. Thus, presence of calcium is obligatory for the production and suppressor activity of SF and it plays a critical role in transmission of the suppressor signal.

Adverse foetal outcome in specific IgM positive Chlamydia trachomatis infection in pregnancy.

A study was undertaken to determine the adverse foetal outcome in antichlamydial IgM positive asymptomatic pregnant females. An indirect immunoperoxidase assay was done to detect IgM in 78 apparently normal asymptomatic pregnant women during the third trimester and follow up was done till delivery to study the effect of chlamydial infection on foetal outcome. A total of 28 (35.9%) women were positive for antichlamydial IgM while only 3.33 per cent asymptomatic non-pregnant normal women (controls) were positive. 28.7 per cent IgM positive mothers delivered low birth weight (LBW) babies (P less than 0.05), 9.7 per cent had premature labour (PTL) and 4.7 per cent had intrauterine death (IUD). The findings are significant as none of the IgM negative mothers had PTL and IUD and only 2.6 per cent had LBW babies.