Surveillance of arbovirus infections in the atlantic forest region, State of São Paulo, Brazil: I. detection of hemagglutination-inhibition antibodies in wild birds between 1978 and 1990

We report data related to arbovirus antibodies detected in wild birds periodically captured from January 1978 to December 1990 in the counties of Salesópolis (Casa Grande Station), Itapetininga and Ribeira Valley, considering the different capture environments. Plasmas were examined using hemagglutination-inhibition (HI) tests. Only monotypic reactions were considered, except for two heterotypic reactions in which a significant difference in titer was observed for a determined virus of the same antigenic group. Among a total of 39,911 birds, 269 birds (0.7%) belonging to 66 species and 22 families were found to have a monotypic reaction for Eastern equine encephalitis (EEE), Venezuelan equine encephalitis (VEE), Western equine encephalitis (WEE), Ilheus (ILH), Rocio (ROC), St. Louis encephalitis (SLE), SP An 71686, or Caraparu (CAR) viruses. Analysis of the data provided information of epidemiologic interest with respect to these agents. Birds with positive serology were distributed among different habitats, with a predominance of unforested habitats. The greatest diversity of positive reactions was observed among species which concentrate in culture fields.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.

Serological diagnosis of dengue by an Elisa inhibition method (EIM)

An ELISA Inhibition Method (EIM) was proposed for the serologic diagnosis of dengue, comparing its results with the Hemagglutination Inhibition (HI) and the IgM capture-ELISA (MAC-ELISA). Advantages and disadvantages of both methods are discussed according to sensitivity, specificity, performance and usefulness. As a conclusion we recommend the complementary inclusion of the EIM and MAC-ELISA substituting the HI for laboratories engaged in the diagnosis and surveillance of dengue.

Serological analysis reveals circulation of influenza C viruses, Brazil

The circulation of influenza C viruses in Rio de Janeiro, Brazil, was studied when significant levels of antibodies were detected (56.7%) with hemagglutination inhibition test, used as a standard methodology for influenza virus studies.

Serologic studies on the behaviour of influenza virus type A in persons of greater São Paulo during 1976, 1978 and 1979

Sera of persons of different age groups collected in 1976, 1978 and 1979 were tested for the presence of HI antibodies against various strains of the H3N2 and H1N1 subtypes of influenza virus. The occurrence of infection by H3N2 subtype was recorded during the 1976-1978 period but in 1979, circulation of this subtype of virus was limited. The prevalence of antibody against A/São Paulo/1/78 (H1N1) was significantly higher than that of antibody to A/USSB/90/77 (HIND in 1978. However in 1979 the predominant strain was A/USSR/90/77 (HIND. Persons under 20 years of age were the most affected by H1N1 subtype. Antibodies to H1N1 subtype were detected in sera of individuals older than 20 years in 1976, before the re-emergence of this strain. Serological results indicate that infections by H3N2 subtype in 1978 occurred in 65.4% of young children (0-4 year group). About 47.0% of children from the 0-4 year group had antibodies to H1N1 subtype in 1979. Antibodies to swine influenza virus were detected in 60% of 60+ year old people.

Plague surveillance in Brazil: 1983 - 1992

Plague caused by Yersinia pestis, has persisted in Brazil in several natural foci spread throughout rural areas in the States of Ceara, Paraiba, Pernambuco, Piaui, Rio Grande do Norte, Alagoas, Bahia, Minas Gerais and Rio de Janeiro. Nationwide surveillance of plague in Brazil based on serological testing started in 1983. We now present an update report of the examinations carried out in our laboratory from 1983 to 1992. The passive hemagglutination test for antibodies against fraction 1A antigen of Y. pestis and the passive hemagglutination inhibition control were employed for testing a total of 220,769 sera. Samples analyzed included 2,856 sera from clinically diagnosed plague cases or suspects, 49,848 sera from rodents of 24 species and 2 species of small wild carnivores (marsupials), 122,890 sera from dogs, and 45,175 sera from cats. Specific antibodies were found in 92 (3.22%) human sera; 143 (0.29%) sera from rodents of 8 species and from the two species of marsupials, 1,105 (0.90%) sera from dogs and 290 (0.64%) sera from cats. The presence of significant levels of specific anti-F1A antibodies among rodents and wild or domestic carnivores (dogs and cats) indicates that all the Brazilian plague foci remain active in spite of the absence of human cases in some of them.

Secondary dengue infection in schoolchildren in a dengue endemic area in the State of Rio de Janeiro, Brazil

A seroepidemiologic survey was carried out in schoolchildren from public schools of the Niterói municipality, state of Rio de Janeiro, Brazil, after a period of sequential epidemics by dengue virus type 1 and 2 (DEN-1 and DEN-2). 450 blood samples were obtained by fingertip puncture and collected on filter paper discs. The hemagglutination inhibition (HAI) test was carried out using DEN-1 and DEN-2 antigens. HAI titres were demonstrated in 66% (297/450) of the sera and the geometric means of the titres were 1/182 and 1/71 for DEN-1 and DEN-2, respectively. Secondary infections were observed in 61% (181/297) of positive cases. Among these, 75% (135/181) were under fifteen years old. No dengue haemorrhagic fever (DHF) was reported in these children. Asymptomatic or oligosymptomatic infections were detected in 56% of the studied population. The absolute and relative frequencies of positive tests by age group and sex did not evidence statistically significant difference. The number of individuals infected probably produced a immunologic barrier responsible for the non occurrence of dengue epidemic in the latter years.

Measles antibody prevalence after mass immunization campaign in Niterói, State of Rio de Janeiro, Brazil

Three months after a mass vaccination campaign (coverage: 100%) against measles a random seroepidemiological survey was carried out in students aged 1 to 19 years old in the Municipality of Niterói, State of Rio de Janeiro. Blood samples were tested for measles antibodies by enzyme immunosorbent assay (EIA) and negative cases were tested again using hemagglutination inhibition (HI) and plaque reduction neutralization (PRN). Of the 798 samples tested by EIA, 718 (90.2%) were positive for measles antibodies. PRN test was more sensitive than EIA and HI in detecting measles specific antibodies. The total antibody prevalence increased from 90.2% to 93.2% when HI was employed in EIA negative specimens and to 98.9% when PRN was used. After the mass vaccination campaign a marked decrease in measles incidence was observed in the municipality studied, showing the effectiveness of the strategy used for measles control in developing countries.

Involvement of the central nervous system in dengue fever: three serologically confirmed cases from Fortaleza, Ceará, Brazil

Three cases of dengue fever involving the central nervous system (CNS) are reported. All occurred in 1994 during a dengue (DEN) epidemic caused by serotypes DEN-1 and DEN-2. The first case examined was a 17-year-old girl who complained of fever, nuchal rigidity and genital bleeding. Three blood samples were positive by anti-dengue IgM ELISA and showed hemagglutination-inhibition (HI) test titers ³ 1,280. The second case concerned a 86-year-old woman with fever, muscle and joint pains, altered consciousness, syncope, nuchal rigidity and meningismus. Her blood sample showed an HI titer of 1:320 for flaviviruses, and an IgM ELISA positive for dengue. The third case was a 67-year-old woman with fever, abnormal behaviour, seizures, tremor of extremities, thrombocytopenia, increased hematocrit and leukopenia. The patient suffered a typical case of dengue hemorrhagic fever with ensuing shock and a fatal outcome. A single blood sample showed HI antibodies of ³ 1,280 and an IgM ELISA positive for dengue. No virus could be isolated from any patient by inoculation of blood into C6/36 cells and suckling mice. No other agent of disease was encountered in the patient.

Outbreak of influenza type A (H1N1) in Iporanga, São Paulo State, Brazil

From June to July 1999 an outbreak of acute respiratory illness occurred in the town of Iporanga. Out of a total of 4,837 inhabitants, 324 cases were notified to the Regional Surveillance Service. Influenza virus was isolated from 57.1% of the collected samples and 100% seroconversion to influenza A (H1N1) was obtained in 20 paired sera tested. The isolates were related to the A/Bayern/07/95 strain (H1N1). The percentages of cases notified during the outbreak were 28.4%, 29.0%, 20.7%, 6.2% and 15.7% in the age groups of 0-4, 5-9, 10-14, 15-19 and older than 20 years, respectively. The highest proportion of positives was observed among children younger than 14 years and no cases were notified in people older than 65 years, none of whom had been recently vaccinated against influenza. These findings suggest a significant vaccine protection against A/Bayern/7/95, the H1 component included in the 1997-98 influenza vaccine for elderly people. This viral strain is antigenically and genetically related to A/Beijing/262/95, the H1 component of the 1999 vaccine. Vaccines containing A/Beijing/262/95 (H1N1) stimulated post-immunization hemagglutination inhibition antibodies equivalent in frequency and titre to both A/Beijing/262/95-like and A/Bayern/7/95-like viruses. Thus, this investigation demonstrates the effectiveness of vaccination against influenza virus in the elderly.

St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

Mayaro virus: imported cases of human infection in São Paulo State, Brazil

Mayaro virus (MAYV) is an arbovirus (Togaviridae: Alphavirus) enzootic in tropical South America and maintained in a sylvan cycle involving wild vertebrates and Haemagogus mosquitoes. MAYV cases occur sporadically in persons with a history of recent activities inside or around forests. This paper reports three cases of MAYV fever detected in men infected in Camapuã, MS, Brazil. Serum samples collected at four days and two months after the onset of the symptoms and examined by hemagglutination inhibition test, revealed monotypic seroconversion to MAYV. Isolation of the virus was obtained from one of the samples by inoculation of the first blood samples into newborn mice. A suspension of the infected mouse brain was inoculated into C6/36 cells culture and the virus was identified by indirect immunofluorescent assay with alphavirus polyclonal antibodies. RT-PCR, performed with RNA extracted from the supernatant of C6/36 infected cells in the presence of alphavirus generic primers as well as specific MAYV primers, confirmed these results. The reported cases illustrate the importance of laboratory confirmation in establishing a correct diagnosis. Clinical symptoms are not always indicative of a disease caused by an arbovirus. Also MAYV causes febrile illness, which may be mistaken for dengue.

Influenza viruses in adult dogs raised in rural and urban areas in the state of São Paulo, Brazil

In 1970, searching for the interspecies transmission of influenza viruses led to the first study on influenza viruses in domestic animals. Birds and mammals, including human beings, are their natural hosts; however, other animals may also play a role in the virus epidemiology. The objective was to investigate the incidence of influenza viruses in adult dogs raised in rural (9, 19.56%) and urban (37, 80.43%) areas in the state of São Paulo, Brazil. Dog serum samples were examined for antibodies to influenza viruses by the hemagglutination inhibition (HI) test using the corresponding antigens from the circulating viruses in Brazil. Dogs from rural areas presented antibodies to influenza A H3N2, and influenza A H7N7 and H3N8. In rural areas, dog sera displayed mean titers as 94.37, 227.88, 168.14, 189.62 HIU/25 µL for subtypes H1N1, H3N2, H7N7, H3N8, respectively. About 84% and 92% of dogs from urban areas exhibited antibodies to human influenza A H1N1 and H3N2, respectively, with statistical difference at p < 0.05 between the mean titers of antibodies to H1N1 and H3N2. About 92% and 100% were positive for H7N7 and H3N8, respectively. In dogs from urban areas, the mean titers of antibodies against influenza A H1N1, H3N2, H7N7 and H3N8, were 213.96, 179.42, 231.76, 231.35 HIU/25 µL respectively. The difference among them was not statistically significant at p > 0.05. In conclusion, these dogs were positive for both human and equine influenza viruses. The present study suggests the first evidence that influenza viruses circulate among dogs in Brazil.

PRESENCE OF RESPIRATORY VIRUSES IN EQUINES IN BRAZIL

Equines are susceptible to respiratory viruses such as influenza and parainfluenza. Respiratory diseases have adversely impacted economies all over the world. This study was intended to determine the presence of influenza and parainfluenza viruses in unvaccinated horses from some regions of the state of São Paulo, Brazil. Blood serum collected from 72 equines of different towns in this state was tested by hemagglutination inhibition test to detect antibodies for both viruses using the corresponding antigens. About 98.6% (71) and 97.2% (70) of the equines responded with antibody protective titers (≥ 80 HIU/25µL) H7N7 and H3N8 subtypes of influenza A viruses, respectively. All horses (72) also responded with protective titers (≥ 80) HIU/25µL against the parainfluenza virus. The difference between mean antibody titers to H7N7 and H3N8 subtypes of influenza A viruses was not statistically significant (p > 0.05). The mean titers for influenza and parainfluenza viruses, on the other hand, showed a statistically significant difference (p < 0.001). These results indicate a better antibody response from equines to parainfluenza 3 virus than to the equine influenza viruses. No statistically significant differences in the responses against H7N7 and H3N8 subtypes of influenza A and parainfluenza 3 viruses were observed according to the gender (female, male) or the age (≤ 2 to 20 years-old) groups. This study provides evidence of the concomitant presence of two subtypes of the equine influenza A (H7N7 and H3N8) viruses and the parainfluenza 3 virus in equines in Brazil. Thus, it is advisable to vaccinate equines against these respiratory viruses.

PREVALENCE OF ANTIBODIES AGAINST INFLUENZA VIRUS IN NON-VACCINATED EQUINES FROM THE BRAZILIAN PANTANAL

The prevalence of antibodies against Equine Influenza Virus (EIV) was determined in 529 equines living on ranches in the municipality of Poconé, Pantanal area of Brazil, by means of the hemagglutination inhibition test, using subtype H3N8 as antigen. The distribution and possible association among positive animal and ranches were evaluated by the chi-square test, spatial autoregressive and multiple linear regression models. The prevalence of antibodies against EIV was estimated at 45.2% (95% CI 30.2 - 61.1%) with titers ranging from 20 to 1,280 HAU. Seropositive equines were found on 92.0% of the surveyed ranches. Equine from non-flooded ranches (66.5%) and negativity in equine infectious anemia virus (EIAV) (61.7%) were associated with antibodies against EIV. No spatial correlation was found among the ranches, but the ones located in non-flooded areas were associated with antibodies against EIV. A negative correlation was found between the prevalence of antibodies against EIV and the presence of EIAV positive animals on the ranches. The high prevalence of antibodies against EIV detected in this study suggests that the virus is circulating among the animals, and this statistical analysis indicates that the movement and aggregation of animals are factors associated to the transmission of the virus in the region.