Dengue virus identification by transmission electron microscopy and molecular methods in fatal dengue hemorrhagic fever.

Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.

Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections.

OBJECTIVES: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue infection were studied. DESIGN: Serum, saliva, and urine samples were collected from 22 patients with clinical and confirmed dengue 3 virus infection during the outbreak in Havana City in 2001. They were tested by capture IgM (MAC-ELISA), IgA (AAC-ELISA), and IgE (EAC-ELISA) and IgG ELISA inhibition method (EIM) to detect specific dengue antibodies. RESULTS: Similar kinetics were observed in IgM, IgA, and IgG antibodies in saliva and IgA and IgG in urine samples from secondary cases compared with kinetics in serum samples, although the values were lower. No IgG antibody was detected in saliva and urine samples in primary cases and IgM antibody was not detected in urine samples from either primary or secondary infection. All secondary cases were positive for IgG in saliva and urine samples at day 7. The kinetics of specific IgE antibodies in primary and secondary cases were different. CONCLUSIONS: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infection were studied for the first time, showing its behavior and usefulness in dengue virus diagnosis. The specific IgE could play a role as a serological marker in secondary infections.

Antibodies from patients with dengue viral infection mediate cellular cytotoxicity.

Acute and late convalescent sera (collected at day 5 of disease onset and 1 year later) from dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) laboratory confirmed cases, were tested for antibody-dependent cell-mediated cytotoxicity (ADCC) activity using dengue 1 (DENV-1) or dengue 2 (DENV-2) infected cells as target. All patients experienced their first dengue virus (DENV) infection 20 years before. ADCC activity was detected in acute sera from DHF/DSS but not in sera from DF patients. However, 1 year after illness, ADCC activity was observed in all cases. This preliminary report represents one of the few studies of ADCC in dengue patients and suggests that ADCC could be implicated in dengue pathogenesis.

Serological markers during dengue 3 primary and secondary infections.

BACKGROUND: The detection of the IgM antibody for the dengue virus in serum by ELISA has become one of the most important and useful methods for diagnosis of dengue using a single acute-phase serum sample. Currently, this system is an invaluable tool for the surveillance of dengue fever (DF) and dengue hemorrhagic fever (DHF). The usefulness of other serological markers such as IgA and IgE have been less studied. OBJECTIVE: To study the IgM, IgA and IgE specific antibody response in dengue 3 infected patients with different clinical picture and type of infection. STUDY DESIGN: One hundred and twenty-seven serum samples collected on days 5-7 at the onset of fever from clinically and serologically confirmed dengue cases were studied. Forty-two were classified as primary dengue fever cases, 48 as secondary dengue fever cases and 37 as secondary dengue hemorrhagic fever cases. All samples were tested by capture ELISA in order to detect dengue IgM, IgA and IgE antibodies. RESULTS AND CONCLUSIONS: In this study, significant differences were observed in the IgM, IgA and IgE response between the study groups. High IgA and IgE OD ratios in secondary dengue cases were found. The usefulness of serotype specific IgM antibody detection is also analyzed and discussed. A priority for future dengue research in terms of protection, recovery of infection and immunopathogenesis is to elucidate the role of these immunoglobulins. The cross reactivity response to IgM between dengue virus serotypes in primary and secondary cases should also be more studied.

Generic RT-nested-PCR for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification.

Flaviviruses are a widespread and numerous group of arboviruses that can cause serious illness in humans. The continuous and slow spread of certain flaviviruses, such as Dengue viruses, and the recent entry and spread of West Nile virus to the American continent, point to the need to control these infections. This control requires the use of suitable techniques for diagnostic and surveillance programmes. A generic RT-nested-PCR that is, theoretically, able to detect each member of the group has been designed. The identification of the detected virus is carried out by sequencing. The introduction of an internal control would reduce the number of false negative results and could be used to quantify the viral load in clinical samples where the method works well.

Monoclonal antibody against Saint Louis encephalitis prM viral protein.

Saint Louis encephalitis virus belongs to Flavivirus genus; Flaviviridae family jointly with other medically important flaviviruses including dengue virus and West Nile virus. The biological properties and functions of prM flavivirus protein are under investigation due to its importance in the generation of infectious virion and host interactions. Monoclonal antibodies have become powerful tools in this approach. Also the use of monoclonal antibodies has been successfully applied for antigenic analysis, clinical diagnosis and treatments. Here, using an immunofluorescence assay we describe a monoclonal antibody (mAb 3D2) that uniquely recognizes native prM Saint Louis encephalitis virus protein expressed in either C6/36-HT or Vero cells. In conclusion, mAb3D2 has significant potential for use in (a) the diagnosis of infections caused by this virus and (b) therapeutic use to treat patients infected by this virus and fundamental research to understand the role of the prM in the Saint Louis encephalitis virus infectious process.

Partial nucleotide and amino acid sequences of the envelope and the envelope/nonstructural protein-1 gene junction of four dengue-2 virus strains isolated during the 1981 Cuban epidemic.

In 1981, an epidemic of dengue hemorrhagic fever (DHF) caused by dengue-2 virus occurred in Cuba. This was the first DHF epidemic reported in the Western Hemisphere. In this study, we have analyzed four dengue-2 Cuban strains for two short genomic fragments: one on the envelope (E) glycoprotein and one at the E/nonstructural protein-1 (NS1) gene junction. The E segment of these 1981 Cuban isolates were more closely related to older dengue-2 virus strains such as New Guinea C 1944, Thailand 1964, Sri Lanka 1968, and Burma 1976 than to more recent isolates of this virus from Jamaica and Vietnam. More than 9% of the divergence with strains isolated from Jamaica and Vietnam was observed at the E/NS1 gene junction. One nucleotide change was observed between the first strain isolated during the epidemic and the rest of the Cuban strains. This mutation induced a nonconserved amino acid change from phenylalanine to leucine at position 43 that was not observed in any of the other strains with which it was compared.

Detection and genetic relationship of dengue virus sequences in seventeen-year-old paraffin-embedded samples from Cuba.

This study describes the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) to generate dengue 2 amplicons from paraffin-embedded autopsy tissues collected in Cuba 17 years ago. The presumptive diagnoses had been made only by clinical evolution without serologic confirmation. This study confirms once again that dengue 2 virus was directly associated with the fatal cases in children and illustrates the potential of the RT-PCR for retrospective diagnosis of dengue cases 17 years after death. A close similarity in the genomic sequences of the dengue 2 RNA detected in tissue samples from fatal cases and those dengue 2 Cuban strains that had been previously investigated confirms the appropriate genomic classification of the etiologic agent associated with the 1981 dengue hemorrhagic fever Cuban epidemic.

Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study.

In Cuba, 2 epidemics of dengue virus occurred: 1 caused by DEN-1 in 1977 and 1 caused by DEN-2 in 1981. The latter was associated with cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). To study viral risk factors for DHF/DSS, a retrospective seroepidemiological survey was conducted in Cerro, a densely populated district in Havana City. The prevalence of plaque reduction neutralizing antibodies to DEN-1 and DEN-2 viruses was measured in 1,295 individuals (children and adults). Of these, 43.7% were immune to DEN-1 virus and 23.6% to DEN-2 virus. Of those individuals who were immune, 26.1% were immune to DEN-1 virus only, 6% to DEN-2 virus only, and 17.6% to both viruses. The DEN-2 virus infection rate in DEN-1 immune individuals was 3.8 times higher than in non-immune individuals. The 5 DHF/DSS cases in the sample had evidence of DEN-1 virus plus DEN-2 virus infections. Three were children and 2 were young adults. No cases were found in individuals infected with DEN-1 virus or DEN-2 virus only. Children infected by DEN-1 virus followed by DEN-2 virus had a high risk of acquiring DHF/DSS. Blacks and whites were equally infected with DEN-1 and DEN-2 viruses.

Why dengue haemorrhagic fever in Cuba? 1. Individual risk factors for dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS).

During the dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) epidemic in Cuba in 1981, we identified some individual risk factors for the development of the severe clinical picture or for the fatal outcome of the disease. The percentage of secondary infection in 3 groups of patients with DHF/DSS was between 95 and 98.3 and it is concluded that secondary infection is an important, but not the only, condition for the development of DHF/DSS. An analysis of these 3 groups of patients and a fourth group of fatal cases showed that chronic diseases such as bronchial asthma, diabetes mellitus and sickle cell anaemia were additional risk factors contributing significantly to the development of DHF/DSS. The study also revealed that race was an individual risk factor, since DHF/DSS was more prevalent in white than in black persons.

Why dengue haemorrhagic fever in Cuba? 2. An integral analysis.

The epidemiological factors present in Cuba in 1981, when the dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) epidemic occurred, were exceptional when compared to those of other countries in the region. Evidence is presented which demonstrates that virulence of the circulating strain is an important element in the analysis of an epidemic. Although the two current hypotheses to explain the occurrence of DHF/DSS epidemics are valid in well defined but different epidemiological situations, neither Halstead's hypothesis of secondary-type infection or Rosen's hypothesis of the role played by the virulence of the circulating strain can explain all cases. An integrated, multifactorial and unifying hypothesis is presented, which could be applied in different epidemiological situations. It is based mainly on an in-depth analysis of the literature and of the Cuban experience.

Dengue haemorrhagic fever in Cuba. I. Serological confirmation of clinical diagnosis.

Serological studies by the haemagglutinin inhibition test to confirm a clinical diagnosis of dengue were done on 406 patients during the dengue 2 epidemic in Cuba in 1981. 49% of the cases were serologically positive; of these 64% was classified as primary and 36% as secondary. The frequency of symptoms is described: the most frequent were fever, headache, malaise and vomiting. Haemorrhagic manifestations predominated significantly in the secondary cases. The white race and female sex were found to predominate in the positive cases.

Dengue haemorrhagic fever in Cuba. II. Clinical investigations.

Clinical and serological studies were carried out on 114 patients admitted to hospital in Havana, Cuba with Dengue Haemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS). Serological confirmation of dengue was obtained in 90% of cases, with 5% of cases primary and 95% secondary. Fever, haemorrhagic manifestations, vomiting and headache were the most frequent signs and symptoms. Among haemorrhagic manifestations, petechiae and vaginal bleeding were reported in a larger number of patients. 21 patients presented shock and, of these, 20 were secondary infections. The disease appeared more frequently in white persons and in women. The aetiopathogenicity of the syndromes is discussed. 95% of the cases could be explained on the basis of the secondary infection hypothesis.

Study of biologic attributes of Cuban dengue 2 virus after serial passage in primary dog kidney cells.

OBJECTIVE: The serial passage of dengue viruses in primary dog kidney (PDK) cells has resulted in selection of attenuated viruses. However, the molecular changes responsible for loss of virulence are not well characterized. This article describes the isolation and biologic attributes of one dengue 2 virulent strain as a first step to allow the study of determinants of virulence at the molecular level. METHODS: A15 dengue 2 Cuban strain was isolated from the viremic plasma of a patient with uncomplicated dengue fever during the 1981 epidemic. This was then subjected to serial passage in PDK cells. Viruses resulting from several PDK passages were compared to the parent strain for plaque size and temperature sensitivity, neurovirulence in newborn mice, and cytopathogenic effects on LLC-MK(2) and C6/36-HT cell lines. RESULTS: A15 dengue 2 Cuban strain was successfully propagated in PDK cells. Primary dog kidney 52 to 53 viruses exhibited several biologic attributes, such as small plaques, temperature sensitivity, reduced mouse neurovirulence, and cytopathic effect in permissive cell lines. CONCLUSIONS: These results represent the first step to allow attenuation of this strain of dengue 2 virus.

Fatal dengue hemorrhagic fever in Cuba, 1997.

OBJECTIVES: After more than 15 years without dengue activity, a dengue II epidemic was reported in Cuba in 1997. Three thousand and twelve serologically confirmed cases were reported, with 205 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases and 12 fatalities. This report presents the clinical, serologic, and virologic findings in the 12 fatal DHF/DSS cases. METHODS: Serum and necropsy samples were studied by viral isolation in C636 cell line and polymerase chain reaction. Serum samples were tested by IgM capture enzyme-linked immunoassay (ELISA) and ELISA inhibition method (EIM). RESULTS: All 12 cases were classified as DHF/DSS according to the Pan American Health Organization Guidelines for Control and Prevention of Dengue and Dengue Hemorrhagic Fever in the Americas. All patients were older than 15 years. Women were more frequently affected. The symptoms and signs presented by these patients were similar to those previously described in DHF/DSS cases. Clinical deterioration occurred on average at day 3.75. Abdominal pain and persistent vomiting were the earliest and most frequent warning signs. Dengue infection was confirmed in all cases. IgM antibodies were detected in 11 of 12 cases, all of them with a secondary infection. Dengue II virus was detected by viral isolation in 12 samples and by polymerase chain reaction in 17. Virus or RNA was detected in various tissues, including kidney, heart, lung, and brain. CONCLUSION: The clinical, pathologic, and laboratory features of 12 cases of fatal dengue hemorrhagic fever were reviewed. The results obtained demonstrate that adults with a primary dengue infection are at risk of developing the severe disease (DHF) if they are infected with a different serotype.

Viral isolation from cases of epidemic neuropathy in Cuba.

OBJECTIVE: To investigate the possibility of a viral agent in the central nervous system of patients with epidemic neuropathy. DESIGN: Virus isolation attempts, in cell cultures and suckling mice, from cerebrospinal fluid (CSF) of neuropathy patients and controls undergoing lumbar puncture for unrelated reasons. Serologic studies in patients, contacts, and controls. SETTING: An epidemic of optic and peripheral neuropathy affected more than 50,000 people in Cuba in 1991 through 1993. Illness was associated with dietary limitations and increased physical demands accompanying the shortages of food and fuel experienced in Cuba since 1989. Most patients responded to parenteral vitamin therapy, and the epidemic began to subside when oral vitamin supplementation was begun for the entire Cuban population. RESULTS: Coxsackievirus A9 (five isolates) and a similar, less cytopathic virus (100 isolates) were recovered from 105 (84%) of 125 CSF specimens from neuropathy patients. The strains with light cytopathic effect were antigenically related to Coxsackieviruses A9 and B4 by cross-neutralization and immunoblotting assays. Virus persisted in CSF of some patients for 1 to 12 months. Cerebrospinal fluid from patients and both types of virus from cell culture produced illness, including complete posterior flaccid paralysis, in newborn mice, and virus was reisolated from the mice. Mouse tissues and sural nerve biopsy specimens from patients were stained by immunoperoxidase and colloidal gold techniques using hyperimmune rabbit antisera against the virus with light cytopathic effect. CONCLUSIONS: Coxsackievirus A9 or an antigenically related agent with a light cytopathic effect was present in CSF of 84% of 125 patients with epidemic neuropathy. The role of these agents, probably in combination with nutritional factors, in the pathophysiology of the disease requires further investigation.

Mechanism of enterovirus involvement in epidemic neuropathy: hypothesis regarding pathophysiology.

During the epidemic of optic and peripheral neuropathy which occurred in Cuba in 1992-1993, viruses antigenically related to the Coxsackie viruses were isolated from cerebrospinal fluid of patients. Concurrently with the virologic studies, epidemiologic, toxicologic, nutritional, immunologic, and histopathologic investigations were also carried out. Although it was demonstrated that the illness was associated with toxic and nutritional risk factors, it has not been possible to identify a specific etiology for the symptoms observed. Taking into consideration the results obtained in all of the various investigations, we have formulated an integral, multifactorial hypothesis which attempts to explain a pathophysiologic mechanism by which the viruses isolated could participate in the pathogenesis of the illness. We propose that the viral agents produce a persistent infection, and the possibility that they may act as mediator of an autoimmune pathogenic process.

[Dengue epidemic in Nicaragua, 1985]. / Epidemia de dengue en Nicaragua, 1985.

In the second half of the year 1985, during the rainy season, an epidemic of Dengue Fever was recognized in Nicaragua. A total of 17,483 cases were reported by the health services. The highest morbidity and attack rates were reported between August and November of the same year. Regions II (Leon and Chinandega), III (Managua) and IV (Masaya, Granada, Carazo y Rivas) reported 89% of the cases; these regions are precisely the more densely populated in the country, located in the Pacific Coast. Leon and Chinandega were the more affected cities reporting 41% of the cases of the epidemic. Sixty seven percent of the cases were adults, 57% were female. The national attack rate was 55.4/10,000 inhab. An intensive campaign for the control of the vector was launched immediately after the epidemic was recognized and by the month of October 1985 morbidity decreased and an endemic situation was established. Seven fatal cases were reported in adults. They were considered as DHF/DSS cases by a multidisciplinary group of pathologists and clinicians, considering the criteria of WHO and the experience obtained during the cuban DHF/DSS epidemic, in 1981. The outbreak was considered as a Classical Dengue Fever epidemic with 7 fatalities. Dengue-1 and -2 were isolated from acute sera and Dengue-1 from one of the dead.

[Mineral composition of milk produced in Monterrey, N. L. Mexico]. / Composición mineral de leche producida en Monterrey, N.L. México.

Milk is a food rich in oligoelements (approx. 1g/100 ml) that can vary in concentration due to genetic factors, type of feed, fed to cattle and due to contamination in the area of production. Since the margin between the concentration of mineral requirements and the toxic levels is so narrow that a little excess results harmful to sensitive species, the present study was done in the city of Monterrey, N.L. located in an industrial zone. Samples of raw milk from 17 producers distributed in 8 zones were analyzed during 6 months, measuring minerals of nutritional and toxicological importance. All elements were determined using atomic absorption, except for phosphorus, which was quantified colorimetrically. Statistical significance was determined through analysis of variance and Duncan test. The average mineral composition of the milk analyzed was (mg/Lt): calcium 1179, magnesium 109.7, zinc 5.89, phosphorus 637.1, copper 0.15, cadmium < 0.01, chromium < 0.02, iron 0.59, lead < 0.04, manganese < 0.02, nickel 0.36. The milk of this region is a good source of calcium, magnesium, phosphorus and zinc, with 35%, 7.4%, 19% and 9% respectively of the RDA being obtained from a 237 ml portion of milk.