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Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading in newer areas in the tropical and subtropical regions of the World. In last fifty years, in spite of intensive and extensive investigations, pathogenesis of dengue is still not clearly understood. Recently, the research focus is on studying the role of intracellular events in pathogenesis of viral infections. Entry of virion in the host cell is followed by quick succession of events, unfolded protein response, lipid bodies and lipophagy, endoplasmic reticulum stress and recent demonstration of autophagy. The turbulence caused by these events may result in clearance of the virus/enhanced replication and survival of the host cell/apoptosis. Both, increased virus load and apoptosis of host cell may have pathological effects on the host. In the present review, we have summed up the role of various intracellular events in viral infections with special emphasis on Dengue virus infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Dengue virus belongs to family Flaviviridae, having four serotypes that spread by the bite of infected Aedes mosquitoes. It causes a wide spectrum of illness from mild asymptomatic illness to severe fatal dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Approximately 2.5 billion people live in dengue-risk regions with about 100 million new cases each year worldwide. The cumulative dengue diseases burden has attained an unprecedented proportion in recent times with sharp increase in the size of human population at risk. Dengue disease presents highly complex pathophysiological, economic and ecologic problems. In India, the first epidemic of clinical dengue-like illness was recorded in Madras (now Chennai) in 1780 and the first virologically proved epidemic of dengue fever (DF) occurred in Calcutta (now Kolkata) and Eastern Coast of India in 1963-1964. During the last 50 years a large number of physicians have treated and described dengue disease in India, but the scientific studies addressing various problems of dengue disease have been carried out at limited number of centres. Achievements of Indian scientists are considerable; however, a lot remain to be achieved for creating an impact. This paper briefly reviews the extent of work done by various groups of scientists in this country.

Dengue virus (DV) infection causes either a benign syndrome, dengue fever, or a severe syndrome, dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS), that is characterized by systemic capillary leakage, thrombocytopaenia and hypovolaemic shock. DHF/DSS occur mainly due to secondary infection by a heterotype DV infection in children and adults but in infants even primary infection by DV causes DHF/DSS. Clinical manifestations of DHF/DSS are more significantly associated with death in infants compared with older children. Vertical transmission of DV and anti-DV IgG has been well reported and is responsible for the pathogenesis of DV disease and its manifestations in infants. The complex pathogenesis of DHF/DSS during primary dengue in infants, with multiple age-related confounding factors, offers unique challenges to investigators. Dengue in infants is not often studied in detail due to practical limitations, but looking at the magnitude of DHF/DSS in infants and the unique opportunities this model provides, there is a need to focus on this problem. This paper reviews existing knowledge on this aspect of DV infection and the challenges it provides.

Advances in free radical research show that reactive oxygen and nitrogen oxide species, for example superoxide, nitric oxide (NO) and peroxynitrite, play an important role in the pathogenesis of different viral infections, including dengue virus. The pathogenic mechanism of dengue haemorrhagic fever (DHF) is complicated and is not clearly understood. The hallmarks of the dengue disease, the antibody-dependent enhancement, the shift from T-helper type 1 (Th1) to Th2 cytokine response and the cytokine tsunami resulting in vascular leakage can now be explained much better with the knowledge gained about NO and peroxynitrite. This paper makes an effort to present a synthesis of the current opinions to explain the pathogenesis of DHF/shock syndrome with NO on centre stage.

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.

Increased vascular permeability without morphological damage to the capillary endothelium is the cardinal feature of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). Extensive plasma leakage in various tissue spaces and serous cavities of the body, including the pleural, pericardial and peritoneal cavities in patients with DHF, may result in profound shock. Among various mechanisms that have been considered include immune complex disease, T-cell-mediated, antibodies cross-reacting with vascular endothelium, enhancing antibodies, complement and its products, various soluble mediators including cytokines, selection of virulent strains and virus virulence, but the most favoured are enhancing antibodies and memory T cells in a secondary infection resulting in cytokine tsunami. Whatever the mechanism, it ultimately targets vascular endothelium (making it a battlefield) leading to severe dengue disease. Extensive recent work has been done in vitro on endothelial cell monolayer models to understand the pathophysiology of vascular endothelium during dengue virus (DV) infection that may be translated to help understand the pathogenesis of DHF/DSS. The present review provides a broad overview of the effects of DV infection and the associated host responses contributing towards alterations in vascular endothelial cell physiology and damage that may be responsible for the DHF/DSS.

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.

Dengue virus was the first microorganism that was shown to induce generation of antigen-specific suppressor T (TS) cells in mice. The cascade of the three generations of TS cells (TS1, TS2, TS3) and their secretary products, the suppressor factors (SF1, SF2), was delineated. The TS pathway was proposed to be protective through inhibition of the production of enhancing antibody, which may enhance the severity of dengue disease. The currently second most favoured mechanism of severe dengue disease is the 'cytokine tsunami'. During the last decade, suppressor/regulatory T cells have been studied in greater detail using modern techniques in various diseases, including viral infections. This brief review discusses the role of dengue-specific suppressor T cells in protection and/or induction of severe dengue disease in view of our current understanding of suppressor/regulatory T cells.

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.

Objective: In order to use facultative gut bacteria as an alternate to animals for the initial gastrointestinal toxicity screening of heavy metals, a comparative study on rat intestinal epithelial cells and resident gut bacteria was undertaken., Methods: in vitro growth rate of four gut bacteria, dehydrogenase (DHA) and esterase (EA) activity test, intestinal epithelial and bacterial cell membrane enzymes and in situ effect of arsenite were analysed., Results: Growth profile of mixed resident population of gut bacteria and pure isolates of Escherichia coli, Pseudomonas sp., Lactobacillus sp., and Staphylococcus sp. revealed an arsenite (2-20 ppm) concentration-dependent inhibition. The viability pattern of epithelial cells also showed similar changes. DHA and EA tests revealed significant inhibition (40%-72%) with arsenite exposure of 5 and 10 ppm in isolated gut bacteria and epithelial cells. Decrease in membrane alkaline phosphatase and Ca2+ -Mg2+ -ATPase activities was in the range of 33%-55% in four bacteria at the arsenite exposure of 10 ppm, whereas it was 60%-65% in intestinal epithelial villus cells. in situ incubation of arsenite using intestinal loops also showed more or less similar changes in membrane enzymes of resident gut bacterial population and epithelial cells., Conclusion: The results indicate that facultative gut bacteria can be used as suitable in vitro model for the preliminary screening of arsenical gastrointestinal cytotoxic effects.

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.

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.

Metal ions are integral part of some viral proteins and play an important role in their survival and pathogenesis. Zinc, magnesium and copper are the commonest metal ion that binds with viral proteins. Metal ions participate in maturation of genomic RNA, activation and catalytic mechanisms, reverse transcription, initial integration process and protection of newly synthesized DNA, inhibition of proton translocation (M2 protein), minus- and plus-strand transfer, enhance nucleic acid annealing, activation of transcription, integration of viral DNA into specific sites and act as a chaperone of nucleic acid. Metal ions are also required for nucleocapsid protein-transactivation response (TAR)-RNA interactions. In certain situations more than one metal ion is required e.g. RNA cleavage by RNase H. This review underscores the importance of metal ions in the survival and pathogenesis of a large group of viruses and studies on structural basis for metal binding should prove useful in the early design and development of viral inhibitors.

The present study was undertaken to investigate the effects of dengue virus (DV) infection in male mice given drinking water containing 250 ppm Cr (VI) and the normal control male mice given plain water to drink. On the basis of intake of water in 24 h, the average dose of Cr (VI) in each mouse was 14.8 mg/kg. After 3, 6, and 9 weeks of drinking Cr (VI), a set of five mice from each group were inoculated intracerebrally (ic) with a 1000 x LD(50) (100 times the lethal dose that kills 50% mice) dose of DV, and the effects on the spleen were studied at the fourth and eightth day postinoculation. It was observed that Cr (VI) drinking and DV infection led to reduction in the weight of the spleen, but the peak reduction was seen in Cr (VI)-fed mice infected with DV, being 30, 34, and 61% at 3, 6, and 9 weeks respectively. A similar response was seen with respect to the cytotoxic activity of spleen homogenates, phagocytic activity of macrophages, and the mitogenic response of spleen cells to concanavalin A from different groups of animals, being most marked (58 to 60%) at the ninth week of Cr (VI) drinking. This shows a summation of adverse effects of DV infection in mice preexposed to Cr (VI).

The major nonoccupational source of chromium (Cr) for humans is through ingestion with food and water, but its effect on the gut microflora has not been studied. The present study was, therefore, undertaken to investigate the effects of chronic ingestion of potassium dichromate (chromium VI) on the resident gut bacteria of male Wistar rats. A group of rats was kept on drinking water containing 10 ppm chromium VI (Cr [VI]) (called Cr-stressed animals) and the other group was given plain water. After 10 weeks, Lactobacillus, Pseudomonas sp., and Escherichia coli were isolated from the cecum of the rats and various studies were performed. The most significant findings of the present study were the stimulation of growth of facultative gut bacteria from the Cr-stressed rats and the significant increase of growth even in the presence of lower concentrations of Cr. Furthermore, the capacity to reduce Cr (VI) was significantly decreased along with the increased tolerance of the bacteria to Cr (higher minimum inhibitory concentration [MIC] values), which was associated with the development of antibiotic resistance. The effects were most marked with the Pseudomonas sp. and least with the E. coli. The antibiotic resistance developed with the Lactobacillus may be a blessing in disguise, because the bacteria may continue to provide benefits even in patients given antibiotic therapy. The gut bacteria thus provide the first line of defense to the body by converting toxic Cr (VI) to a less toxic Cr (III) and may act as a prebiotic.

Toxicants including heavy metals reaching the intestine following ingestion through food and water primarily interact with an ecosystem of eukaryotic and prokaryotic cells. Gut bacteria having a dynamic interrelationship with intestinal epithelial cells are known to play important and specific metabolic, trophic, and protective functions. The present study was undertaken to compare the effects of hexavalent chromium on rat intestinal epithelial cells and the resident gut bacteria following in vitro and in vivo exposures. The survival rate and viability pattern of two types of cells were comparable. Under in vitro conditions, the gut bacteria were quick to reduce Cr (VI) in early time periods, while, at 30 h time, both types of cells showed similar capacity for the reduction of Cr (VI). Chromium intoxication (10 ppm of Cr (VI) in drinking water for 10 weeks) caused significant decrease in membrane alkaline phosphatase and Ca(2 +)-Mg(2 +)-ATPase activities of intestinal epithelial cells as well as of three gut bacteria viz. Escherichia coli, Pseudomonas sp, and Lactobacillus sp. Major structural membrane constituents like carbohydrates and phospholipids also showed significant decline in both types of cells. These findings indicate that 10 ppm and higher Cr concentrations may cause toxic insult, resulting in impaired intestinal functional efficacy. It also implies that the gut bacteria can be used at least for preliminary screening of heavy metals gastrointestinal toxicity.

The present study was undertaken to investigate the spectrum of bacteria present in the River Gomti water before and after chlorination for drinking purposes. We observed that the strains of Pseudomonas aeruginosa that survived chlorination on three out of seven occasions were resistant to almost all the antibiotics tested. The chlorine-resistant bacteria had mucoid colonies and grew better at 24 degrees C. All attempts to isolate the plasmid responsible for chlorine resistance were unsuccessful. Laboratory experiments using different strains of the P. aeruginosa in distilled water showed that only the resistant strain survived chlorine treatment at a dose of < or =500 microg/L. Similar results were obtained when water collected from seven different sites on the River Gomti was treated with graded doses of chlorine. At the higher dose of chlorine, all the bacteria died in 30 min, whereas with lower doses all the bacteria survived. The present study underscores the importance of measuring water chlorine concentrations to assure they are sufficiently high to remove pathogenic bacteria from drinking water. To our knowledge, this is the first report in the literature of the selection of multidrug-resistant bacteria by suboptimal chlorine treatment of water.

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.

Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mphi) to produce a biologically active cytotoxic cytokine, the Mphi cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mphi. The present study was undertaken to identify the CF2-receptors (CF2-R) on murine T cells followed by their purification and characterization. Receptor binding assay and Scatchard analysis revealed single, high-affinity (1.0309 nM) receptors for CF2 on T cells (22000 receptors per cell). The binding of [125I]CF2 on murine T cells was saturable and specific. Furthermore, CF2-R was purified from normal mouse T cell plasma membrane by affinity chromatography followed by reversed-phase high-pressure liquid chromatography. The presence of CF2-R was confirmed by indirect dot-blot assay and its binding with [125I]CF2. The purified CF2-R is a 90-95-kDa protein as characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. The chemical crosslinking of [125I]CF2 and its receptor complex showed a product of 100-110 kDa on different subpopulations of murine T cells. The pretreatment of target cells with anti-CF2-R antisera inhibited the cytotoxic activity of CF2 in a dose-dependent manner and thus confirmed the biological significance of CF2-R. Moreover, the presence of CF2-R was also identified on normal human peripheral blood mononuclear cells and T and B cells by crosslinking with [125I]CF2, thus revealing the possible role of CF2 and CF2-R in the immunopathogenesis of dengue virus disease.

The cells of the immune system form a strong line of defence against foreign substances. The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro. 5 x 10(6) cells were incubated with 10 or 25 microg ml(-1) of Cr (VI) in the form of K2Cr2O7 at 37 degrees C in the presence of 5% CO2 in air. At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated. Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages. The reduction by splenic and peritoneal macrophages was similar. Total thymocytes reduced 54% of the Cr (VI). Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated. Among the intestinal cells the maximum reduction of 100% (of 10 microg ml(-1)) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells. The reduction in the intestinal loop in situ was 100%. The time taken by each cell type for the peak reduction to Cr (VI) was markedly different. The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium. The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects.

Diseases caused by dengue, sandfly fever and hanta viruses pose a major health risk in many countries. We determined the threat of these arboviral infections through a serologic using enzyme linked immunosorbent assay (ELISA) based tests. Hantavirus-specific antibodies were also detected using immunofluorescence. Of 499 samples tested for dengue virus IgG antibodies l4% were as positive for dengue positive by all the ELISA tests. Among the 42 showing strong IgG reactivity, only 1 was positive for dengue virus IgM antibodies. All samples tested for IgG antibodies to sandfly fever virus were negative. Hantavirus antibodies were detected in 11% of the 46 samples from high-risk individuals. The low prevalences suggest that at present these infections are not a serious problem in Kuwait.

Chromium is a naturally occurring heavy metal found commonly in the environment in trivalent, Cr(III), and hexavalent, Cr(VI), forms. Cr(VI) compounds have been declared as a potent occupational carcinogen among workers in chrome plating, stainless steel, and pigment industries. The reduction of Cr(VI) to Cr(III) results in the formation of reactive intermediates that together with oxidative stress 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. On the other hand, chromium is an essential nutrient required to promote the action of insulin in body tissues so that the body can use sugars, proteins and fats. Chromium is of significant importance in altering the immune response by immunostimulatory or immunosuppressive processes as shown by its effects on T and B lymphocytes, macrophages, cytokine production and the immune response that may induce hypersensitivity reactions. This review gives an overview of the effects of chromium on the immune system of the body., (Copyright 2002 Federation of European Microbiological Societies)

Hantaviruses are found in widely scattered areas of the world and are transmitted by inhalation of virus-contaminated aerosols of rodent excreta. The present study was undertaken in Kuwait to investigate the serological evidence for hantavirus infection in rodents and humans. Sera were collected from 283 wild rodents and 183 human subjects (46 Kuwaitis and 137 non-Kuwaitis). The rodent sera were investigated for the presence of antibodies against the Seoul and Puumala strains of the hantaviruses by enzyme-linked immunosorbent assay and immunofluorescence technique using the virus-infected Vero E6 cells. The findings showed the presence of anti-hantavirus antibodies in seven out of the 283 (2.8%) rodents. Antibodies against the Seoul strain were present in six (2.1%) and against the Puumala strain in three (1%) rodents. Further, it was observed that three out of 84 (3.6%) of the Rattus norvegicus and four out of 174 (2.3%) Mus musculus had anti-hantavirus antibodies. Two rodents belonging to species Mus musculus had antibodies against both strains of the hantaviruses. Out of 183 human sera, 13 (7%) were positive for hantavirus antibodies. Among the Kuwaitis 5/46 (11%) and among the non-Kuwaitis 8/137 (6%) were positive for the hantavirus antibodies. Antibodies to both Puumala and Hantaan strains were detected in Kuwaitis as well as in non-Kuwaitis. Although no human case of hantavirus illness has yet been reported in Kuwait, the serological evidence of infection suggests a constant vigil.

This study was carried out on sera from 210 patients in Kuwait in 1997-1999. All of the patients were suffering from febrile illness and had recently visited dengue- (DEN) endemic areas. The sera were screened for DEN virus by inoculation into cultures of the Aedes albopictus cell clone C6/36 (virus isolation) and by IgM capture ELISA (detection of DEN virus-specific IgM antibodies). In the cell cultures, DEN virus could not be isolated from any of the patients' sera. However, sera from 19 patients were positive for DEN virus-specific IgM antibodies. All these 19 sera were tested for the presence of DEN virus-specific RNA by reverse transcription-PCR (RT-PCR) using DEN virus types-common (consensus) primers. In addition, the type of DEN virus was identified by using type-specific primers in a semi-nested PCR. The results showed that two of the 19 patients were infected with DEN virus type 2. This report of 19 patients with serological evidence of DEN infection indicates that imported DEN is a real threat to Kuwait, a country non-endemic for DEN but with a large portion of the population vacationing in DEN-hyperendemic areas during the peak DEN season and then returning to Kuwait.

Interleukin (IL)-13 is produced by T helper 2 (Th2)-type cells and inhibits the production of proinflammatory cytokines by activated monocytes, while IL-18 is a pleiotropic cytokine that induces interferon-gamma and plays an important role in the development of Th1-type cells. Role of the shift from a Th1-type response to Th2-type has been suggested in the pathogenesis of dengue hemorrhagic fever (DHF). This study was undertaken to investigate the possible protective/pathogenic role of IL-13 and IL-18 in patients with DHF. Sera were collected from a total of 84 patients with various grades of dengue illness and 21 normal healthy controls and tested for IL-13 and IL-18 levels using commercial enzyme-linked immunosorbent assay kits. The results showed that very low levels of IL-13 (4+/-3 pg ml(-1)) and IL-18 (15+/-4 pg ml(-1)) were detected in the sera of healthy controls. In dengue patients, the levels of IL-13 and IL-18 were the highest in the patients with DHF grade IV (205+/-103 pg ml(-1) and 366+/-155 pg ml(-1), respectively) and the lowest in patients with dengue fever (22+/-12 pg ml(-1) and 76+/-50 pg ml(-1), respectively). Both the cytokines appeared (IL-13=20+/-11 pg ml(-1) and IL-18=70+/-45 pg ml(-1)) during the first 4 days of illness and reached peak levels (IL-13=204+/-96 pg ml(-1) and IL-18=360+/-148 pg ml(-1)) by day 9 onwards. The presence of high levels of IL-13 and IL-18 during severe illness and late phases of the disease suggests that both of these cytokines may contribute to the shift from a Th1- to Th2-type response and thus to the pathogenesis of DHF.

During dengue virus infection a unique cytokine, cytotoxic factor (hCF), is produced that is pathogenesis-related and plays a key role in the development of dengue haemorrhagic fever (DHF). However, what regulates the adverse effects of hCF is not known. We have previously shown that anti-hCF antibodies raised in mice, neutralise the pathogenic effects of hCF. In this study we have investigated the presence and levels of hCF-autoantibodies in sera of patients with various severity of dengue illness (n=136) and normal healthy controls (n=50). The highest levels of hCF-autoantibodies (mean+/-S.D.=36+/-20 U ml(-1)) were seen in patients with mild illness, the dengue fever (DF), and 48 out of 50 (96%) of the sera were positive. On the other hand the hCF-autoantibody levels declined sharply with the development of DHF and the levels were lowest in patients with DHF grade IV (mean+/-S.D.=5+/-2 U ml(-1); P=<0.001 as compared to DF). Only one of the 13 DHF grade IV patients had an antibody level above the 'cut-off' value (mean plus 3 S.D. of the control sera). The analysis of data with respect to different days of illness further showed that the highest levels of hCF-autoantibodies were present in DF patients at >9 days of illness. Moreover, the DF patients at all time points, i.e. 1-4, 5-8 and >9 days of illness had significantly higher levels of hCF-autoantibodies (P<0.001) than patients with DHF grade I, II, III and IV. In addition DHF grade I and grade II patients had significantly more positive specimens than DHF grade III and grade IV patients at all time points. These results suggest that elevated levels of hCF-autoantibodies protect the patients against the development of severe forms of DHF and, therefore, it may be useful as a prognostic indicator.

Dengue virus produces a mild acute febrile illness, dengue fever (DF) and a severe illness, dengue hemorrhagic fever (DHF). The characteristic feature of DHF is increased capillary permeability leading to extensive plasma leakage in serous cavities resulting in shock. The pathogenesis of DHF is not fully understood. This paper presents a cascade of cytokines, that in our view, may lead to DHF. The main feature is the early generation of a unique cytokine, human cytotoxic factor (hCF) that initiates a series of events leading to a shift from Th1-type response in mild illness to a Th2-type response resulting in severe DHF. The shift from Th1 to Th2 is regulated by the relative levels of interferon-gamma and interleukin (IL)-10 and between IL-12 and transforming growth factor-beta, which showed an inverse relationship in patients with DF.

Interleukin (IL)-12 has a broad range of activities including regulation of cytokine synthesis and selective promotion of Th1-type cell development. A shift from a Th1-type response to Th2-type has been suggested to be important in the pathogenesis of dengue hemorrhagic fever (DHF). This study was undertaken to investigate the possible role of IL-12 in this shift. A total of 76 patients with various grades of dengue illness and 21 normal healthy controls were tested for IL-12 levels in serum samples and IL-12 mRNA in their peripheral blood mononuclear cells. The results showed that the levels of IL-12 were the highest in patients with dengue fever (270+/-102 pg ml(-1)) followed by decreasing levels in the patients with DHF grade I (198+/-86 pg ml(-1); P<0.05) and DHF grade II (84+/-52 pg ml(-1); P<0.001). Neither IL-12 nor its mRNA could be detected in the patients with DHF grades III and IV. The cytokine appeared and reached peak levels during the first 4 days of illness, started to decline by day 5-8 and disappeared by day 9 onwards. The absence of IL-12 during severe illness and late phases of the disease may be responsible for the shift to a Th2-type response and thus for the pathogenesis of DHF.

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.

The study was undertaken to elucidate the sequence of appearance of T helper (Th)1- and Th2-type cytokines in human peripheral blood leucocyte cultures infected in vitro with dengue type 2 virus. Commercial sandwich enzyme-linked immunosorbent assay kits were used to assay the levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, and IL-10 in culture supernatants. Culture supernatants were also screened for the cytotoxic factor and the dengue virus titres determined. The cytokines that appeared in the culture supernatants on the first day post-infection (p.i.) were cytotoxic factor, TNF-alpha, IL-2, and IL-6; their levels were highest on the second day p.i. IFN-gamma appeared on the second day with a peak on the third day p.i. The levels of these cytokines declined quickly, except for human cytotoxic factor (hCF) and IL-2. The cytokines that appeared later were IL-10 and IL-5 on the fourth day and IL-4 on the sixth day p.i. Dengue virus replicated in the peripheral blood leucocyte (PBL) cultures and was present throughout the course of the study. The findings of the present study show that dengue virus induced a predominant Th1-type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2-type response later.

The pathogenesis of dengue haemorrhagic fever (DHF) is incompletely understood but it has been suggested that various cytokines may have a role in the process. In this study the profile of the cytokine Transforming Growth Factor-beta 1 (TGF-beta1) was investigated in the sera of 79 patients with various grades of dengue illness and in 21 normal healthy controls. Also, TGF-beta1-specific mRNA was examined in their peripheral blood mononuclear cells (PBMC). The results showed that neither TGF-beta1 protein nor its mRNA were detected in healthy controls. In dengue patients, the TGF-beta1 protein and its mRNA were detected in 96%. However, among the patient groups, the levels of TGF-beta1 were lowest in patients with dengue fever (DF; mean value 315 +/- 95 pg/ml) and were highest in patients with DHF grade IV (mean value 1350 +/- 280 pg/ml; P = < 0. 001). The cytokine appeared during the first four days of illness (304 +/- 90 pg/ml) and gradually increased, reaching peak levels (1050 +/- 215 pg/ml) after the 9th day of the illness. Thus TGF-beta1 in the sera and TGF-beta1-mRNA in the PBMC were present in most of the patients with dengue (96%) but the cytokine levels were highest during the later periods of illness and in patients with DHF grade IV, suggesting a possible role of TGF-beta1 in the pathogenesis of DHF.

Dengue virus causes dengue fever, a mild febrile illness, and at times dengue hemorrhagic fever (DHF), a severe illness the pathogenesis of which is not fully understood. Given the crucial roles played by interleukin-8 (IL-8) as a chemoattractant cytokine and in inflammatory processes, levels of circulating IL-8 in the sera and IL-8 mRNA in the peripheral blood mononuclear cells (PBMC) were measured in 99 patients of a recent dengue epidemic that occurred in India in 1996 and in 21 normal healthy controls. Twenty-six of the patients had dengue fever (DF) and the remaining 73 were diagnosed as having different grades of DHF. All the control normal sera were negative for IL-8, so were their PBMC for IL-8 mRNA. Increased levels of IL-8 in the sera and IL-8 mRNA in their PBMC were observed in patients with severe illness of DHF grades III and IV. Only two out of 26 patients of DF and one out of 10 DHF grade I patient were positive for IL-8 and all three deteriorated to DHF grade IV within 24 hr. All six patients of DHF grade IV who died had higher serum level of IL-8 above 200 pg/ml, the highest being 5,568 pg/ml in one patient; the presence of mRNA for IL-8 was very high in all patients. A striking correlation was observed between increased levels of IL-8 and severe DHF, with greater levels in patients with increased grade of the disease and death. These results suggest that IL-8 may have an important role and may be an indicator of increasing severity of the disease and death.

A unique cytokine, human cytotoxic factor (hCF), has been shown to occur in the sera of patients with dengue fever (DF) and dengue haemorrhagic fever (DHF). The present study was undertaken to investigate the ability of fresh PBMC of such patients to produce hCF. The PBMC were cultured for 24 h and the culture supernatants (CS) were analysed for the presence of hCF by cytotoxicity assay, competitive ELISA and dot blot tests. In 90% of 246 cases CS were positive for hCF by the three tests. CS were positive for hCF in PBMC collected from days 1-20 of illness but not at later periods. Higher cytotoxic activity was observed in CS of days 1-4 of illness and was highest in cases of DHF grade IV and lowest in cases of DF. Dot blot hybridization of RNA extracted from the PBMC of the patients showed the presence of mRNA for hCF in 94% of cases. A similar number of patients showed the presence of hCF in situ in the PBMC smears by fluorescent antibody technique. hCF was found only in CD4+ T cells. The findings thus present direct evidence of the production of hCF by CD4 T cells of cases of DF/DHF.

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.

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 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.

We report here development of hypoglycaemia in the convalescent phase of Japanese encephalitis virus (JEV) infection in mice by the induction of antigen-specific Ly1- 2+ T cells in the spleen which mediate hypoglycaemia through the generation of soluble T cell hypoglycaemic factor (TCHF). The TCHF acted in a dose-dependent manner and was found to be trypsin-sensitive and thermolabile. It was purified on Superose-12 high performance liquid chromatography (HPLC) gel filtration column and purified protein migrated as a approximately 25-kD band on SDS-PAGE. The JEV-induced hypoglycaemia coincided with an increased circulating glucagon level, without any alterations in blood insulin and growth hormone concentrations. These effects were mimicked by TCHF. These results indicate that JEV-primed T lymphocytes mediate hypoglycaemia through the production of a soluble hypoglycaemic factor.

Dengue type 2 virus (DV) induces a subpopulation of T lymphocytes of mice to produce a cytokine, cytotoxic factor (mCF), which induces H-2A positive macrophages to produce macrophage cytotoxin (CF2). The present study was undertaken to investigate the mechanism of cytotoxicity of CF2. It was observed that CF2 induced production of superoxide anion (O2-) and hydrogen peroxide (H2O2) by the spleen cells of mice in vitro and in vivo. The maximum production of O2- (260 +/- 10 nM/4 x 10(6) cells) was at 45 minutes while that of H2O2 was at 90 minutes after inoculation of CF2. Pretreatment of mice or spleen cells with anti-CF2-antisera inhibited O2- and H2O2 production in a dose-dependent manner. Superoxide dismutase (SOD) inhibited O2- production and cytotoxicity while H2O2 production was increased by increasing SOD concentration in the culture. This indicated that O2- production is necessary for the cytotoxic activity of CF2. Pretreatment of the cells with Ca2+ channel blocking drugs, nifedipine or verapamil, inhibited CF2-induced O2- and H2O2 production in a dose-dependent manner. We have shown earlier that the cytotoxic activity of CF2 is known to be Ca2+ dependent and CF2-induced production of nitrite and the cytotoxicity is inhibited by NG-monomethyl-L-arginine. Thus, it is suggested that O2- and nitrite are necessary for cell killing by CF2 in a Ca(2+)-dependent manner and the killing may possibly be by generation of peroxynitrite.

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.

Dengue type 2 virus (DV) infection induces production of a cytokine, the cytotoxic factor (CF) in the spleen of mice. The present study was undertaken to investigate the production of nitrite (NO2-) by the spleen cells of mice in vitro and in vivo following inoculation of DV or CF. Maximum NO2- production occurred at 45 min after inoculation of 5 microg CF, both in vitro and in vivo. The NO2- was produced by macrophages and T cells and not by B cells. Pretreatment of CF with anti-CF antisera inhibited production of NO2-. DV-stimulated spleen cell culture supernatants showed peak production of CF and NO2- at 72h. In DV-infected mouse spleen, maximum NO2- production occurred at 8-11 days post-infection, which correlated with peak cytotoxic activity in the spleen. Pretreatment of spleen cells with N(G)-monomethyl L-arginine (NMMA) inhibited NO2- production. NO2- production was abrogated in a dose-dependent manner by treatment of spleen cells with Ca2+ channel blocking drug, Nifedipine. The findings demonstrate that DV-induced CF induces production of NO2- in spleen cells, probably in a Ca2+-dependent manner, and may be a mechanism of target cell killing.

Dengue type-2 virus (DV) infection in mice induces T cells to produce a cytokine, the cytotoxic factor (CF), which induces H2-A positive macrophages to produce another cytokine, cyototoxic (CF2), which amplifies its cytotoxic effects on target cells. The present study was undertaken to investigate the production of nitrite (NO2-) by the spleen cells of mice in vitro and in vivo following inoculation of CF2. Maximum NO2- production occurred at 1 hour after inoculation of 100 micrograms CF2. Pretreatment of CF2 with anti-CF2-antisera (CF2-As) inhibited the production of NO2-. Pretreatment of the spleen cells with NG-monomethyl-L-arginine (NMA) or with arginase inhibited NO2- production. The NO2- production was diminished in a dose dependent manner by treatment of spleen cells with the Ca2+ channel blocking drug, nifedipine and Zn2+ as ZnSO4. The findings of the present study thus demonstrate that CF2 induces production of NO2- in the spleen cells in a CA(2+)-dependent manner which may be a mechanism of target cell killing.

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.

Specific active immunization with dengue type 2 virus (DV)-induced cytokine, cytotoxic factor (CF), prevents CF-mediated pathology in mice. The present study was undertaken to determine the optimum dose of CF and the effect of different adjuvants on the immune response as assessed by the study of anti-CF antibody titre by ELISA and protection against increase in capillary permeability to challenging dose of 3 micrograms CF. The maximum protection of 94 +/- 4% against increase in capillary permeability was observed at week 4 after immunization with 5 micrograms dose of CF mixed with Freund's incomplete adjuvant (FIA), which gradually decreased to 21 +/- 10% on week 24. With a dose of 10 micrograms the protection obtained was 79 +/- 5%, but persisted for a longer time at a higher level. The response was poor with 1 microgram dose of CF. The mean anti-CF antibody titres gradually decreased after reaching the peak at week 4 after immunization. Mice immunized with different adjuvants emulsified with 5 micrograms CF were challenged at different intervals with 3 micrograms CF. Maximum protection observed with CF + tetanus toxoid (TT) and 84/246 was about 93 +/- 2% and 97 +/- 2%, while that with alhydrogel was 33 +/- 12% and with bacille Calmette-Guérin (BCG) was 67 +/- 4%. At week 24 after immunization, however, the best response was obtained with 10 micrograms of adjuvant 84/246. Intracerebral challenge with 10 or 100 LD50 dose of dengue type 2 virus showed significantly prolonged mean survival time and delayed onset of signs of sickness in immunized mice compared with normal mice. The maximum survival time was with adjuvant 84/246 even at week 24. The findings thus show that the optimum dose of CF is 5 micrograms and the adjuvant of choice is 84/246.

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.