Re-emergence of rabies virus maintained by canid populations in Paraguay.

Paraguay has registered no human cases of rabies since 2004, and the last case in dogs, reported in 2009, was due to a variant maintained in the common vampire bat "Desmodus rotundus". In 2014, a dog was diagnosed as positive for rabies with aggression towards a boy and all required measures of control were successfully adopted. Epidemiological investigation revealed that the dog was not vaccinated and had been attacked by a crab-eating fox, "zorro" (Cerdocyon thous). The sample was diagnosed by the Official Veterinary Service of the Country and sent to the Center on Rabies Research from the University of São Paulo, Brazil, for antigenic and genetic characterization. A second sample from a dog positive for rabies in the same region in 2015 and 11 samples from a rabies outbreak from Asuncion in 1996 were also characterized. The antigenic profile of the samples, AgV2, was compatible with one of the variants maintained by dogs in Latin America. In genetic characterization, the samples segregated in the canine (domestic and wild species)-related group in an independent subgroup that also included samples from Argentina. These results and the epidemiology of the case indicate that even with the control of rabies in domestic animals, the virus can still circulate in wildlife and may be transmitted to domestic animals and humans, demonstrating the importance of continuous and improved surveillance and control of rabies, including in wild species, to prevent outbreaks in controlled areas.

The emergence of wildlife species as a source of human rabies infection in Brazil.

Forty-five human rabies virus isolates from a wide geographical area of Brazil were characterized using an anti-nucleoprotein monoclonal antibody panel and by partial nucleotide sequencing analysis of the nucleoprotein gene. Three major antigenic groups related to the antigenic variants maintained in domestic dogs, vampire bats and marmosets were identified. Phylogenetic analyses revealed that the viruses from dog-related cases segregated into four sister clades: three associated with dog-endemic cycles in Brazil and one with the crab-eating fox cycle in the northeastern region of the country. The vampire bat- and marmoset-related viruses formed two independent groups. The topology of these clades was conserved when these samples were compared to virus representatives of the currently reported rabies endemic cycles in the Americas. These results indicated the presence of multiple endemic transmission cycles maintained in four different reservoirs, domestic dogs, crab-eating foxes, vampire bats and marmosets, which are being transmitted directly to humans and should be considered as a high-risk for rabies infection.

Epidemiological and molecular characterization of rubella virus isolated in São Paulo, Brazil during 1997-2004.

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997-2004 were isolated in cell culture and genotyped. Twenty-eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil.

Phylogenetic analysis of rubella virus strains during an outbreak in São Paulo, 2007-2008.

Rubella virus (RV) is an important human pathogen that causes rubella, an acute contagious disease. It also causes severe birth defects collectively known as congenital rubella syndrome when infection occurs during the first trimester of pregnancy. Here, we present the phylogenetic analysis of RV that circulated in São Paulo during the 2007-2008 outbreak. Samples collected from patients diagnosed with rubella were isolated in cell culture and sequenced. RV RNA was obtained from samples or RV-infected cell cultures and amplified by reverse transcriptase-polymerase chain reaction. Sequences were assigned to genotypes by phylogenetic analysis using RV reference sequences. Seventeen sequences were analyzed, and three genotypes were identified: 1a, 1G, and 2B. Genotypes 1a and 1G, which were isolated in 2007, were responsible for sporadic rubella cases in São Paulo. Thereafter, in late 2007, the epidemiological conditions changed, resulting in a large RV outbreak with the clear dominance of genotype 2B. The results of this study provide new approaches for monitoring the progress of elimination of rubella from São Paulo, Brazil.

Detection of hantaviruses in Brazilian rodents by SYBR-Green-based real-time RT-PCR.

Current knowledge of the pathogenic hantavirus indicates that wild rodents are its primary natural reservoir. Specific primers to detect the presence of viral genomes were developed using an SYBR-Green-based real-time RT-PCR protocol. One hundred sixty-four rodents native to the Atlantic Forest biome were captured in São Paulo State, Brazil, and their tissues were tested. The presence of hantavirus RNA was detected in sixteen rodents: three specimens of Akodon montensis, three of Akodon cursor, two of Necromys lasiurus, one of Juliomys sp., one of Thaptomys nigrita, five of Oligoryzomys nigripes, and one of Oryzomys sp. This SYBR Green real-time RT-PCR method for detection of hantavirus may be useful for surveying hantaviruses in Brazil.

Hospital-acquired human bocavirus in infants.

Human bocavirus (HBoV) is a respiratory pathogen that affects young children. We screened 511 nasopharyngeal aspirates for hospital-acquired HBoV from infants hospitalised with respiratory infection from January to December 2008. Among 55 children with HBoV infection, 10 cases were hospital-acquired. Compared with the community-acquired cases, coinfection with other respiratory viruses in these patients was uncommon. HBoV should be considered for inclusion in screening protocols for nosocomial childhood respiratory infections, especially in intensive care units.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples.

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Isolation and genotype analysis of rubella virus from a case of Guillain-Barré syndrome.

Rubella virus (RV) infection has sporadically been linked to Guillain-Barré syndrome (GBS), but the association with RV has been based only on clinical and/or serological backgrounds. In the present case it was possible to isolate RV (genotype 1a) from cerebrospinal fluid and peripheral blood mononuclear cells of an 18-year-old woman diagnosed with GBS after clinical manifestations of rubella. This report contributes to confirm RV as one of the triggering pathogens of this peripheral nervous system disease.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0 percent for sensitivity and 91.2 percent for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0 percent and 91.2 percent, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0 percent, 89.0 percent, 84.0 percent and 99.0 percent, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5 percent for sensitivity and 95.4 percent for specificity, as well as PPV and NPV of 92.9 percent and 86.0 percent, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Seroprevalence of human parvovirus B19 in a suburban population in São Paulo, Brazil

OBJETIVO: Analisar a prevalência de anticorpos IgG ao parvovírus humano B19. MÉTODOS: Estudo transversal em uma comunidade de subúrbio de São Paulo, Brasil, de novembro 1990 a janeiro de 1991. Amostras aleatórias (N=435) e representativas de soro foram coletadas de crianças sadias a partir de 15 dias de idade e de adultos com até 40 anos. Os anticorpos IgG ao parvovírus humano B19 foram detectados pelo teste ELISA. RESULTADOS: A prevalência de anticorpos IgG ao parvovírus B19 foi de 87 por cento dos recém-nascidos. A prevalência de anticorpos IgG de origem materna decaiu exponencialmente até o 19o mês de idade. Baixa prevalência de anticorpos foi observada nos primeiros quatro anos de vida, aumentando até 72 por cento no grupo etário de 31-40 anos. A idade média de aquisição da primeira infecção nesta comunidade é de 21 ± 7 anos. A idade ótima para se vacinar as crianças desta comunidade com uma vacina hipotética é de um ano de idade. CONCLUSÕES: A prevalência de anticorpos IgG ao parvovírus B19 foi alta entre recém-nascidos e no grupo etário 31-40 anos. A análise por estrutura etária mostrou padrão similar aos estudos prévios relacionados à baixa prevalência de infecção em crianças que aumenta com a idade.

Seroprevalence of human parvovirus B19 in a suburban population in São Paulo, Brazil.

OBJECTIVE: To analyze the prevalence of IgG antibodies to human parvovirus B19. METHODS: Cross-sectional study in a suburban community in São Paulo, Southeastern Brazil, between November 1990 and January 1991. Randomly selected (N=435) representative samples of sera were collected from healthy children older than 15 days old and adults up to 40 years old. IgG antibodies were detected using ELISA. RESULTS: High prevalence of IgG antibodies to B19 parvovirus was found in 87% of newborns. The prevalence of maternally derived IgG antibodies exponentially plunged up to the 19th month of age. Low prevalence of antibodies was found in the first 4 years of life, increasing up to 72% in those aged 31-40 years. It was estimated that the average age of first infection in this population is 21 +/- 7 years old and the optimal age for vaccination with a hypothetical vaccine would be 1 year of age. CONCLUSIONS: Parvovirus B19 IgG antibody prevalence was high in newborns and those aged 31-40 years. The analysis by age groups showed a pattern similar to that found in previous studies, i.e., low prevalence of infection in children that increases with age.

Construction of a recombinant plasmid pSH-G containing the rabies-virus glycoprotein G gene

A plasmid named pSH-G was constructed with the rabies-virus G-gene insert. This plasmid was transfected into eukaryotic BHK-21 cells and its stability tested. The presence of the pSH-G plasmid was confirmed by means of polymerase chain reaction (PCR) after each of ten cell passages, and the results were positive. The stable BHK-21/pSH-G+ clone obtained can be used in the study of rabies as well as in the production of vaccines.

Experimental rabies infection in haematophagous bats Desmodus rotundus.

In order to determine the susceptibility and serum neutralizing antibody response of Desmodus rotundus to rabies virus, bats were inoculated with a virus isolated from a naturally infected haematophagous bat. Bats were divided into four groups of 10 animals each. Dilutions of rabies virus containing 100, 1000, 10,000 and 100,000 MICLD50 (lethal dose 50% for mice inoculated by the intracerebral route) were administrated in the pectoral muscle. The presence of rabies virus was detected in brain and salivary glands by fluorescent antibody, mouse inoculation and RT-PCR. The observed mortality for each virus dose was 0, 20, 20 and 60% respectively. Serum neutralizing antibodies were tested for by the rapid fluorescent focus inhibition test, and antibody titres greater than 0.5 IU/ml were found in 53% of bats 30 days after virus inoculation. Resistance to infection was seen in bats that developed low or no detectable antibody response as well as in bats with high titres. Among the 10 bats that died of rabies, eight showed signs of paralytic rabies and two bats showed no clinical signs.

Molecular analysis of a Measles virus isolate from Brazil: a case originating in Japan.

This study reports on molecular analysis of a Measles virus (MV) isolate from a patient who was infected in Japan but showed symptoms after arriving to Brazil. This patient had typical clinical measles infection symptoms: fever, rash, cough and coryza. After isolating the virus in B95a cells, a fragment of the nucleoprotein (N) gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subjected to direct nucleotide sequencing. The sequence data showed that the MV isolate of concern is of the D5 genotype.

Detection of influenza, parainfluenza, adenovirus and respiratory syncytial virus during asthma attacks in children older than 2 years old.

BACKGROUND: Viral upper respiratory tract infections (URTI) have been correlated with the onset of asthma attacks in children and viral identification was reported in 14-49 % of nasal samples. The aim of the present study was to detect influenza, parainfluenza, adenovirus and respiratory syncytial virus (RSV) in older children during acute asthma attacks. METHODS: A total of 104 children (2-14 years) were included in four groups: group I: asthmatics with acute attack and URTI; group II: asthmatics without URTI (group I children, 30 days later); group III: non-asthmatics with URTI; group IV: non-asthmatic, asymptomatic children. A diagnosis of URTI was considered when (3 symptoms (cough and/or sneeze, nasal obstruction, hypertrophy of turbinates, pain and/or retropharynx hyperemia, headache and fever) in asthmatics and at least 2 symptoms in non-asthmatics were present, starting within 7 days. Samples of nasal mucosa cells (n = 123) were collected, and culture and indirect immunofluorescence were carried out to identify respiratory syncytial virus, adenovirus, influenza A and B, parainfluenza 1,2 and 3 and rhinovirus. RESULTS: Viral identification rates were higher in the asthmatic groups: 13.9 % in group I, 11.1 % in group II; 2.8 % in group III and 0 in group IV. The following viruses were identified: RSV 2/36, rhinovirus 1/36, adenovirus 1/36 and parainfluenzae 1/36 in group I; adenovirus 2/18 in group II; RSV 1/36 in group III. CONCLUSIONS: The rate of viral identification was higher in asthmatic children, whether symptomatic or not, suggesting a possible susceptibility to viral infections. Virus could also be a triggering factor in attacks, although it is not the most preponderant in older children.

Immune response to respiratory syncytial virus in young Brazilian children.

We have evaluated the cellular and humoral immune response to primary respiratory syncytial virus (RSV) infection in young infants. Serum specimens from 65 patients <=12 months of age (39 males and 26 females, 28 cases <3 months and 37 cases > or = 3 months; median 3 3.9 months) were tested for anti-RSV IgG and IgG subclass antibodies by EIA. Flow cytometry was used to characterize cell surface markers expressed on peripheral blood mononuclear cells (PBMC) from 29 RSV-infected children. There was a low rate of seroconversion in children <3 months of age, whose acute-phase PBMC were mostly T lymphocytes (63.0 +/- 9.0%). In contrast, a higher rate of seroconversion was observed in children >3 months of age, with predominance of B lymphocytes (71.0 +/- 17.7%). Stimulation of PBMC with RSV (2 x 10(5) TCID50) for 48 h did not induce a detectable increase in intracellular cytokines and only a few showed a detectable increase in RSV-specific secreted cytokines. These data suggest that age is an important factor affecting the infants' ability to develop an immune response to RSV.

Immune response to respiratory syncytial virus in young Brazilian children

We have evaluated the cellular and humoral immune response to primary respiratory syncytial virus (RSV) infection in young infants. Serum specimens from 65 patients <=12 months of age (39 males and 26 females, 28 cases <3 months and 37 cases > or = 3 months; median 3 ± 3.9 months) were tested for anti-RSV IgG and IgG subclass antibodies by EIA. Flow cytometry was used to characterize cell surface markers expressed on peripheral blood mononuclear cells (PBMC) from 29 RSV-infected children. There was a low rate of seroconversion in children <3 months of age, whose acute-phase PBMC were mostly T lymphocytes (63.0 ± 9.0 percent). In contrast, a higher rate of seroconversion was observed in children >3 months of age, with predominance of B lymphocytes (71.0 ± 17.7 percent). Stimulation of PBMC with RSV (2 x 10(5) TCID50) for 48 h did not induce a detectable increase in intracellular cytokines and only a few showed a detectable increase in RSV-specific secreted cytokines. These data suggest that age is an important factor affecting the infants' ability to develop an immune response to RSV

Detection of human parvovirus B19 in a patient with hepatitis

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3 percent) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis

Detection of human parvovirus B19 in a patient with hepatitis.

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3%) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis.