Rachna Nagar

Elevated levels of IL-8 in dengue hemorrhagic fever.

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. J. Med. Virol. 56:280–285, 1998.

Sequential production of cytokines by dengue virus-infected human peripheral blood leukocyte cultures

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‐α), interferon‐gamma (IFN‐γ), 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‐α, IL‐2, and IL‐6; their levels were highest on the second day p.i. IFN‐γ 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. J. Med. Virol. 59:335–340, 1999.

Dengue and dengue haemorrhagic fever: Indian perspective

The relationship of this country with dengue has been long and intense. The first recorded epidemic of clinically dengue-like illness occurred at Madras in 1780 and the dengue virus was isolated for the first time almost simultaneously in Japan and Calcutta in 1943–1944. After the first virologically proved epidemic of dengue fever along the East Coast of India in 1963–1964, it spread to allover the country. The first 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.

Profile of transforming growth factor-beta 1 in patients with dengue haemorrhagic fever

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‐β1) was investigated in the sera of 79 patients with various grades of dengue illness and in 21 normal healthy controls. Also, TGF‐β1‐specific mRNA was examined in their peripheral blood mononuclear cells (PBMC). The results showed that neither TGF‐β1 protein nor its mRNA were detected in healthy controls. In dengue patients, the TGF‐β1 protein and its mRNA were detected in 96%. However, among the patient groups, the levels of TGF‐β1 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‐β1 in the sera and TGF‐β1‐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‐β1 in the pathogenesis of DHF.

Effect of dengue virus infection on Fc-receptor functions of mouse macrophages

Fc-receptor-mediated attachment and ingestion of opsonized sheep erythrocytes (EA) by the macrophages of spleen and peritoneal cavity were studied during dengue virus type 2 (DV) infection of Swiss albino mice. Following intracerebral inoculation, virus antigen could be demonstrated by immunofluorescence in the splenic macrophages from day 4 and in peritoneal macrophages from day 5 post-infection, with a higher number of positive cells discernible on the 7th and 8th days. The virus could be isolated from spleen tissue from day 5. The total number of cells was markedly reduced from day 4 onwards both in the spleen and peritoneal cavity. A loss in the capacity to attach and ingest EA was noticed, the lowest values of attachment index (AI) and phagocytic index (PI) being reached on day 4. At later periods the AI values increased markedly but continued to be significantly less than those in uninfected control mice. The PI values continued to be lower throughout. The dichotomy between the Fc-mediated attachment and ingestion may be a mechanism for prevention of virus infection of macrophages.

Production of cytotoxic factor by peripheral blood mononuclear cells (PBMC) in patients with dengue haemorrhagic fever

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.

Nitric oxide in dengue and dengue haemorrhagic fever: necessity or nuisance?

Abstract 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.

Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area: co-infection or cross reactivity?

OBJECTIVE To report high co-positivity of anti-dengue virus (DV) and anti-Japanese encephalitis virus (JEV) IgM in an area endemic for both the viruses and to discuss the possibilities of co-infection. METHODS Serum samples from the patients who presented with fever, suspected central nervous system infection and thrombocytopenia, were tested for anti-DV IgM and anti-JEV IgM antibodies. Conventional reverse transcriptase polymerase chain reaction was done for detection of DV RNA and JEV RNA. RESULTS Of 1 410 patient sera tested for anti-DV and anti-JEV antibodies, 129 (9.14%) were co-positive for both. This co-positivity was observed only in those months when anti-JEV IgM positivity was high. Titers of both anti-DV IgM and anti-JEV IgM were high in most of the co-positive cases. Among these 129 co-positive cases, 76 were tested by conventional reverse transcriptase polymerase chain reaction for both flaviviruses, of which eight cases were co-positive for DV and JEV. CONCLUSIONS Co-infection with more than one flavivirus species can occur in hyperendemic areas.

Cytotoxic Factor in Dengue Haemorrhagic Fever

Objective: Cytotoxic factor is a unique pathogenesis-associated cytokine that is produced in mice (mCF) and man (hCF) during dengue virus infection. This study was undertaken to investigate the prevalence of hCF and its relationship to the duration and severity of the illness, and to ascertain its role, if any, in the pathogenesis of dengue fever (DF) and dengue haemorrhagic fever (DHF). Methods: Peripheral venous blood was collected from the patients of various grades and on different days after the onset of clinical illness. Sera were collected from a total of 333 cases, and analysed for the presence of hCF by inhibition ELISA and dot blot tests. Result: The positivity for hCF was 100% in cases of DHF grades III and IV, while overall positivity was seen in 295 out of 333 (88%) cases studied. Sera collected from the 1st to the 20th day of illness were positive for hCF. This was not seen at later periods. A majority of cases (52%) were below 15 years of age and peak positivity of 96% was noticed in the age-group of 11–15 years. The mean inhibition value of the ELISA was lowest (40 ± 8%) in cases of DF and was highest (70 ± 10%) in DHF grade IV, and the peak titres were found on the first 4 days of the illness. Conclusion: The findings show the presence of hCF in the majority of cases. It is detectable up to the 20th day of illness and may suggest an association of higher levels of hCF with the onset and severity of the illness.