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.

ABSTRACT The possibility of a correlation between dengue virus genotype groups and disease severity is currently under discussion. The objective of this investigation was to identify any immunogenic difference between the American and Asian dengue 2 virus genotypes through the study of antibody development (virus-binding immunoglobulin G and neutralizing antibodies) in mice. Differences in the neutralization pattern between the strains studied were observed, suggesting the presence of slight antigenic variations among them. The lack of recognition of one of the Asian genotype strains was remarkable.

Background: During the 1994–1995 season, an increase in the circulation of influenza type B was observed in our infantile population, according to serological studies (hemagglutination-inhibition). We reported the case of four children (<1 year old) that were diagnosed with influenza-like illness, who developed a severe disease (rapid progressive encephalopathy) with high fever, drowsiness and recurrent convulsions, and were presumptively diagnosed with encephalomyocarditis. Brain and lung necropsies were carried out. Materials and methods: Initial laboratory diagnosis was done by immunofluorescence assay on brain and lung samples, followed by viral isolation in MDCK cells and chicken eggs. The isolates were characterized antigenically by haemagglutination-inhibition assay (HI) with post-infection ferret antisera. Results: For the immunofluorescence technique, four samples were positive. In MDCK and chicken eggs, six haemagglutinating agents characterized by hemagglutination-inhibition were similar to the B/Beijing/184/93-like reference strain (four from the lungs and two from the brain). Data from the morbidity report will be presented. Serological studies (monosera and pair sera) showed an increase in the circulation of influenza B in our infantile population. Conclusions: The pathogenesis of encephalopathy is still not clarified. However, we believe that viremia is the key to initiation. There is an urgent need to promote vaccination against influenza in young children to prevent these devastating disease conditions.

We compared a multiplex polymerase chain reaction assay and a shell vial assay for the detection of herpesviruses infection in 13 Cuban patients who had received kidney transplants. Cytomegalovirus and human herpesvirus 6 were detected in these patients.

From April to September 2000, an epidemic of aseptic meningitis spread throughout Cuba, with 16,943 reported cases. Virologic studies identified echovirus 16 as the cause of this epidemic. This is the first reported isolate of echovirus 16 from patients with viral meningitis in Cuba.

Background: In Cuba, flu is the first cause of infectious diseases and the fifth general cause of death associated with pneumonia. Lung necropsies were carried out on 15 fatal cases, between 20–54 years old, and dengue was presumptively diagnosed during an epidemic outbreak in 1997. According to virological evaluation, it was demonstrated that the causative agent in nine of these cases was influenza virus. Techniques and Methods: Indirect Immunofluorescence (IFI); immunoperoxidase staining (IPS); RT-PCR; nucleotide sequence. Culture was in MDCK cells and chicken eggs and hemagglutination inhibition was used for antigenic characterization. Results: With the IFA technique, nine cases (60%) were positive and with IPS, seven cases were positive (46.7%). In MDCK and chicken eggs, six hemagglutinating agents characterized by hemagglutination inhibition were similar to the reference strain A/Johannesburg/33/94 (H3N2). Type and subtype A(H3N2) were demonstrated by RT-PCR, too. Nucleotide sequences were determined and compared. Serological studies showed that the isolated A/Santiago de Cuba/193/97 strain was circulating in the population studied. Morbidity reports during January to May showed an average of 19 600 cases per month; however, morbidity during June and July rose to 39 800 per month. Conclusion: This is the first report about the co-circulation of influenza and dengue viruses in Cuba. It also reinforces the necessity of developing prophylactic, clinical and epidemiological actions against flu during dengue virus epidemics.

A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results suggest that the rapid centrifugation assay may be useful for detection of dengue virus in clinical specimens.

Strains A-15 (isolated in Cuba, 1981), Jamaica (isolated in Jamaica, 1981) and Nueva Guinea "C" (standard) from dengue-2 virus were compared according to the time of appearance of the cytopathic effect (CPE), to the time of appearance of specific fluorescence and to the kynetics of viral multiplication on being innoculated in the cell lines AP-61 (Aedes pseudoscutellaris) and C6/36 HT (Aedes albopictus). The results showed that the CPE of highest intensity and earliest appearance was for A-15, followed by Jamaica and Nueva Guinea "C" (NGC). AP-61 seems to favor the CPE of Jamaica with respect to that of the same strain in C6/36 HT. The fluorescence was earlier for Jamaica and A-15 and more intensive for the latter, whereas NGC manifested late. This behaviour was similar in the 2 cellular systems. The greatest titres during the kinetics of viral multiplication were obtained from A-15 in both lines, although in AP-61 they tend to be equal from the 4th day on. The strain A-15 showed a particular behaviour of these biological properties on comparing them with the other strains under study, which may be related to changes found in its neucleotide sequence.

The antigenic structure and antimicrobial susceptibility were studied in 99 strains isolated from patients with acute diarrhea (6 strains from an outbreak of digestive transmission disease in Santiago de Cuba) and a strain isolated from a patient who died from infections neurological syndrome (INS, meningitis). Four new serotypes (093, 994, 095, 096), which had not been described in the world classification, were identified from the Cuban isolated strains and were included in the International Serotyping Scheme by the International Reference Center located in Prague, Czech Republic. For the first time in Cuba, the circulation of serotypes 017:H11, 011: H2, 023. H1alc, 057: H3 which show cross reaction to Shiguella species was proved. Those strains from the outbreak of digestive disease belonged to serotype 050: H11 and had a thermostable toxin. The first case of infectious neurologic syndrome with Plesionomas shigelloides etiology reported in Cuba was described; the strain corresponded to serotype 050: H11. The worldwide reported pattern of antimicrobial resistance was demonstrated.

The paper presents the case of a female patient who was admitted to "Calixto García" General Hospital with respiratory distress and hypovolemic or septic shock. She was diagnosed with viral hemorrhagic pneumonia. From the endotracheal secretion taken as a sample, the influenza virus was isolated as etiological agent, which, through the hemaglutination inhibition technique, was characterized as a strain belonging to H3N2 subtype, very similar to strain A/Johannesburg/33/94 from the antigenic viewpoint. The patient recovered satisfactorily after being treated with rivabirin.

Some biological properties of Dengue-2 strains such as A-15 (isolated in Cuba in 1981); Jamaica (isolated in Jamaica in 1981) and New Guinea "C" (NG"C") standard strain differing in their nucleotide sequences were studied. The results showed that the cytopathic effect in C6/36 HT cell line occurred earlier in A-15 strain and that fluorescence was first detected in Jamaica and A-15 strains. This seems to indicate that rapid detection of strains does not have any relation to neither their history of passage nor the original isolation system. A-15 and NG"C" strains exhibited an heterogeneous pattern formed by big and small plaques but average size of plaques in NG"C" was lower whereas Jamaica showed only small plaques. The most neurovirulent strain in mice was NG"G" followed by A-15 whereas Jamaica was not neurovirulent at all. These results indicate that A-15 has a different biological behaviour which is probably due to intrinsic differences. It should be taken into account that 7 amino acid changes were found in the envelope protein which may have affected the expression of some biological properties.

We performed an study to find out the main virulence factor in verotoxigenic Escherichia coli: the production of verotoxins in 50 non sorbitol-fermenting Escherichia coli strains (possible enterohemorrhage) which were isolated from children with acute diarrheas in the City of Havana and referred to the National Reference Laboratory for acute diarrheal diseases in "Pedro Kourí" Institute. By using the agglutination technique with latex particles of E. coli O157:H7, we determined whether the verotoxins belonged to this serotype, we also researched the production of verotoxins in Vero cell culture. Ninety-six percent of the total number of strains were positive in the qualitative determination of this factor which was more frequently observed after 24 hours.

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

The NCI-H292 continual line of mucoepidermoid cells of the human lungs has been reported to be useful for the propagation of many viruses, mainly Adenovirus and Paramyxovirus. It is stated the possible substitution of primary cultures of monkey kidney for NCI-H292 in order to isolate such agents. In the present paper it is evaluated the utility of this line for multiplying the respiratory syncytial viruses Adenovirus 3 and 7, and the parainfluenza viruses 1, 2, and 3, in comparison with the continual cellular lines traditionally used for the propagation of these viruses, whose strains were inoculated this time in the Vero, HEp-2, and HeLa lines, according to their know sensitivities as well as in NCI-H292 simultaneously. The viral multiplication was detected by the appearance of the cytopathic effect or by hemadsorption. As a result, it was demonstrated the multiplication capacity of the NCI-H292 line for Adenoviruses 3 and 7 and parainfluenza 3, being more useful for their multiplication than the traditionally used lines.

An ultramicro ELISA assay of double antibody for the detection of IgG antibodies to the respiratory syncytial virus (RSV) was standardized. It was used a RVS antiprotein F monoclonal antibody produced by the Genetic Engineering and Biotechnology Center (GEBC) in Havana. The use of this antibody allowed to include crude antigenic preparations instead of purified fractions, which caused a significant reduction of the reactivity obtained with the antigen control. The assay conditions were determined by crossed titration. It was obtained a sensitivity of 97.2%, a coincidence of 91%, and a specificity of 83.3% of the UMELISA as regards the complement fixation. The results may be qualitatively expressed or by antibody titres using only one serum dilution (1:40) and a pattern curve.

The ELISA was standardized to detect monoclonal antibodies of dengue virus proteins E and NS1. One indirect ELISA was applied, using C6-36 cells inoculated with the A-15 strain, isolated during the dengue 2 epidemic in 1981 as an antigen source. These cells were fixed in ELISA plates at a 200,000 cell/well concentration. A cell control in similar conditions was used. Specific monoclonal antibodies to both proteins were used to standardize the system. Studies at different incubation periods, to determine the highest expression moment of these proteins in the cell membrane, were carried out. The results show a full response at 72 hours postinoculation for both proteins; a 14.7 ng/mL sensitivity was obtained for the detection of NS1, and of 1.43 ng/mL for E protein. This system allows the monoclonal antibodies primary screening to dengue 2 virus E and NS1 proteins.

This paper reports on the necessary conditions for plaque development in mild cutopathogenic effect-producing agents which were isolated from samples of the cerebrospinal fluid of patients presenting with epidemic neuropathy.

The presence of 2 agents such as a Cox A9 strain and another mild cytopathogenic effect-producing strain, both isolated from patients presenting with epidemic neuropathy is reported in this paper. A mild cutopathogenic effect which was propagated in successive dilutions was developed in the dilution 10(-4) by means of the neutralization test of a Coxsackie A9 virus with its homologous hyperimmune serum. A gradient in saccharose was performed in a mild cytopathogenic effect-producing strain and a typical cytopathogenic effect of an Enterovirus developed from one of the fractions passed in tissular cultures in the presence phosphonoacetic acid (PAA). The possible pathogenic role of these viruses are discussed.