Respiratory virus infections in hospitalized and non-hospitalized children: determinants of severe course of the disease.

INTRODUCTION: Viral respiratory disease constitutes a great burden worldwide mainly among children. OBJECTIVE: One pursued to compare disease characteristics of children who required hospitalization from those who did not require hospitalization due to a viral respiratory disease. METHODOLOGY: Medical and demographic data were collected through questionnaires and nasopharyngeal aspirates were tested for detection of respiratory disease viruses of in and outpatients up to five years old, presenting acute respiratory infection. RESULTS: Respiratory syncytial virus predominated among hospitalized children while other viruses (Human rhinovirus, Influenza virus, Parainfluenza virus, Adenovirus, and Human metapneumovirus) together predominated among non-hospitalized patients. Although children with underlying risk condition required longer hospitalization, previously healthy children presented severe disease and required hospitalization as well. Also, clinical characteristics were not found that may distinguish RSV infected children who had comorbidities from those previously healthy. CONCLUSIONS: Children who were hospitalized due to respiratory distress had well defined characteristics: early age, respiratory syncytial virus infection, bronchiolitis and presence of comorbidity. Nevertheless, rapid respiratory syncytial virus identification among early age children may be of great value in order to avoid medical misconduct, such as unnecessary antibiotic prescription and preventive health care before an eventual clinical worsening encompassing previous health status.

Molecular characterization of influenza viruses collected from young children in Uberlandia, Brazil - from 2001 to 2010.

BACKGROUND: Influenza remains a major health problem due to the seasonal epidemics that occur every year caused by the emergence of new influenza virus strains. Hemagglutinin (HA) and neuraminidase (NA) glycoproteins are under selective pressure and subjected to frequent changes by antigenic drift. Therefore, our main objective was to investigate the influenza cases in Uberlândia city, Midwestern Brazil, in order to monitor the appearance of new viral strains, despite the availability of a prophylactic vaccine. METHODS: Nasopharyngeal samples were collected from 605 children less than five years of age presenting with acute respiratory disease and tested by immunofluorescence assay (IFA) for detection of adenovirus, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3 and influenza virus types A and B. A reverse transcription-PCR (RT-PCR) for influenza viruses A and B was carried out to amplify partial segments of the HA and NA genes. The nucleotide sequences were analyzed and compared with sequences of the virus strains of the vaccine available in the same year of sample collection. RESULTS: Forty samples (6.6%) were tested positive for influenza virus by IFA and RT-PCR, with 39 samples containing virus of type A and one of type B. By RT-PCR, the type A viruses were further characterized in subtypes H3N2, H1N2 and H1N1 (41.0%, 17.9%, and 2.6%, respectively). Deduced amino acid sequence analysis of the partial hemagglutinin sequence compared to sequences from vaccine strains, revealed that all strains found in Uberlândia had variations in the antigenic sites. The sequences of the receptor binding sites were preserved, although substitutions with similar amino acids were observed in few cases. The neuraminidase sequences did not show significant changes. All the H3 isolates detected in the 2001-2003 period had drifted from vaccine strain, unlike the isolates of the 2004-2007 period. CONCLUSIONS: These results suggest that the seasonal influenza vaccine effectiveness could be reduced because of A H3N2 variants that circulated in 2001-2003 years. Thus, an early monitoring of variants circulating in the country or in a region may provide important information about the probable efficacy of the vaccine that will be administered in an influenza season.

Influence of the CCR-5/MIP-1 α axis in the pathogenesis of Rocio virus encephalitis in a mouse model.

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5-/- mice, while MIP-1 α-/- mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection.

Hepatitis C virus antigenic convergence.

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development.

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent.

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3% (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI.

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3 percent (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI.

Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus.

Human adenoviruses (HAdV) are a major cause of acute respiratory diseases (ARD), gastroenteritis, conjunctivitis and urinary infections. Between November 2000-April 2007, a total of 468 nasopharyngeal aspirate samples were collected from children with ARD at the Clinics Hospital of Uberlândia. These samples were tested by immunofluorescence assay (IFA) and 3% (14/468) tested positive for the presence of HAdV. By performing polymerase chain reaction (PCR) to detect HAdV DNA in samples that tested negative or inconclusive for all viruses identifiable by IFA (respiratory syncytial virus, parainfluenza viruses 1, 2 and 3, influenza viruses A and B and HAdV), as well as negative for rhinoviruses by reverse transcription-PCR, additional 19 cases were detected, for a total of 33 (7.1%) HAdV-positive samples. Nucleotide sequences of 13 HAdV samples were analyzed, revealing that they belonged to species B, C and E. Further analyses showed that species C (HAdV-2) was the most prevalent among the sequenced samples. To our knowledge, this is the first report describing the presence of HAdV-4 in Brazil. We also detected an isolate that was 100% identical to a part of the feline adenovirus hexon gene sequence.

Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus

Human adenoviruses (HAdV) are a major cause of acute respiratory diseases (ARD), gastroenteritis, conjunctivitis and urinary infections. Between November 2000-April 2007, a total of 468 nasopharyngeal aspirate samples were collected from children with ARD at the Clinics Hospital of Uberlândia. These samples were tested by immunofluorescence assay (IFA) and 3 percent (14/468) tested positive for the presence of HAdV. By performing polymerase chain reaction (PCR) to detect HAdV DNA in samples that tested negative or inconclusive for all viruses identifiable by IFA (respiratory syncytial virus, parainfluenza viruses 1, 2 and 3, influenza viruses A and B and HAdV), as well as negative for rhinoviruses by reverse transcription-PCR, additional 19 cases were detected, for a total of 33 (7.1 percent) HAdV-positive samples. Nucleotide sequences of 13 HAdV samples were analyzed, revealing that they belonged to species B, C and E. Further analyses showed that species C (HAdV-2) was the most prevalent among the sequenced samples. To our knowledge, this is the first report describing the presence of HAdV-4 in Brazil. We also detected an isolate that was 100 percent identical to a part of the feline adenovirus hexon gene sequence.

Hepatitis B virus genotyping among chronic hepatitis B patients with resistance to treatment with lamivudine in the City of Ribeirão Preto, State of São Paulo / Genotipagem do vírus da hepatite B de pacientes crônicos com resistência ao tratamento com lamivudina na Cidade de Ribeirão Preto, Estado de São Paulo

INTRODUCTION: Lamivudine is a nucleoside analogue that is used clinically for treating chronic hepatitis B infection. However, the main problem with prolonged use of lamivudine is the development of viral resistance to the treatment. Mutations in the YMDD motif of the hepatitis B virus DNA polymerase gene have been associated with resistance to drug therapy. So far, there have not been many studies in Brazil reporting on genotype-dependent development of resistance to lamivudine. Thus, the aim of the present study was to determine the possible correlation between a certain genotype and increased development of resistance to lamivudine among chronic hepatitis B patients. METHODS: HBV DNA in samples from 50 patients under lamivudine treatment was amplified by means of conventional PCR. Samples were collected at Hospital das Clínicas, FMRP-USP. The products were then sequenced and phylogenetic analysis was performed. RESULTS: Phylogenetic analysis revealed that 29 (58 percent) patients were infected with genotype D, 20 (40 percent) with genotype A and one (2 percent) with genotype F. Mutations in the YMDD motif occurred in 20 percent of the patients with genotype A and 27.6 percent of the patients with genotype D. CONCLUSIONS: Despite the small number of samples, our results indicated that mutations in the YMDD motif were 1.38 times more frequent in genotype D than in genotype A.

INTRODUÇÃO: Lamivudina é um análogo de nucleosídeo clinicamente utilizado para o tratamento da infecção crônica pela hepatite B. Entretanto, o principal problema do uso prolongado da lamivudina é o desenvolvimento de resistência viral ao tratamento. Mutações no motivo YMDD no gene da DNA polimerase do vírus da hepatite B estão associados com a resistência a terapia medicamentosa. Até o presente momento, não há muitos estudos no Brasil que descrevem o desenvolvimento genótipo-dependente da resistência à lamivudina. Assim, o intuito do trabalho aqui descrito foi determinar a possível correlação entre um determinado genótipo e o desenvolvimento aumentado da resistência à lamivudina em pacientes com hepatite B crônica. MÉTODOS: O HBV DNA foi amplificado por PCR convencional a partir de 50 amostras coletadas de pacientes submetidos ao tratamento com lamivudina no Hospital das Clínicas- FMRP- USP. Posteriormente, os produtos foram seqüenciados e a análise filogenética foi realizada. RESULTADOS: A análise filogenética mostrou que 29 (58 por cento) pacientes foram infectados com o genótipo D, 20 (40 por cento) com o genótipo A e 1 (2 por cento) com o genótipo F. Mutações no motivo YMDD ocorreu em 20 por cento dos pacientes com genótipo A e 27,6 por cento em pacientes do genótipo D. CONCLUSÕES: Apesar do baixo número de amostras, nossos resultados indicaram que mutações no motivo YMDD são 1,38 X mais frequentes no genótipo D em relação ao genótipo A.

Molecular characterization of swine hepatitis E virus from Southeastern Brazil

Despite serological evidences of presence of hepatitis E virus (HEV) in humans or other animals, until this study the virus had not been detected and molecular characterization of the isolate that circulates in Brazil had not been described. Thus, we collected stool samples of young pigs and tested for presence of HEV RNA by RT-PCR, using primers for partial amplification of ORF2 sequence. Phylogenetic analysis with sequence obtained from the amplified products revealed that the HEV isolate identified here was most closely related to HEV isolates of genotype 3, which is commonly detected in HEV infected pigs. Nucleotide sequence analyses carried out with the entire amplified fragment, ORF2/ORF3 overlapping and ORF2 non-overlapping sequences showed highest identities with the US isolate of genotype 3. Similarly, amino acid sequence analyses done with the entire amplified fragment, ORF2 non-overlapping, ORF2 and ORF3 overlapping sequences also showed highest identities with the genotype 3 isolate. Presence, in Brazil, of HEV of genotype 4, which also infects pigs, as well as HEV strains that infect humans still remain to be detected and characterized.

Apesar de evidências sorológicas da presença do vírus da hepatite E (VHE) em humanos ou outros animais, até o presente estudo o vírus não havia sido detectado e a caracterização molecular da cepa que circula no Brasil não havia sido descrita. Assim, amostras de fezes de porcos jovens foram colhidas e analizadas quanto a presença do ARN do VHE por RT-PCR, utilizando-se oligonucleotídeos para amplificação da seqüência parcial do ORF2 viral. Análise filogenética realizada com a seqüência obtida dos produtos amplificados revelou que a cepa encontrada apresentou relação próxima com cepas do genótipo 3 que são detectadas com freqüência em porcos. Análises das seqüências nucleotídicas realizadas com todo o segmento amplificado, com somente o segmento sobreposto do ORF2/ORF3 e aquele sem sobreposição do ORF2, em comparação com isolados de genótipos conhecidos, mostraram maior identidade com o isolado encontrado nos Estados Unidos (US) do genótipo 3. De maneira semelhante, análises da seqüência de aminoácidos realizadas com os mesmos segmentos também mostraram maior identidade com o isolado de genótipo 3. Presença ou não do vírus de genótipo 4, que também infecta porcos, ainda necessita ser verificada. Da mesma forma, cepas do VHE que infectam humanos ainda necessitam ser detectadas e caracterizadas.

Clinical-epidemiological evaluation of respiratory syncytial virus infection in children attended in a public hospital in midwestern Brazil

Respiratory syncytial virus (RSV) is responsible for annual respiratory infection outbreaks in infants and young children worldwide, frequently causing bronchiolitis and pneumonia. We evaluated clinical and epidemiological features of acute respiratory infections (ARIs) caused by respiratory syncytial virus (RSV) in children less than five years old. Nasopharyngeal aspirate samples from children with ARI symptoms, attended at the 'Hospital das Clínicas' - Federal University of Uberlândia, MG, Brazil, were collected and tested for RSV by the immunofluorescence assay (IFA). Patients' clinical and epidemiological data were also obtained. From April 2000 to June 2003, 317 nasopharyngeal samples were collected from children less than 54 months old. Seventy-six samples (24.0 percent) were positive for RSV, with 53 percent (40/76) obtained from male patients. Hospitalization occurred in 50 percent (38/76) of the cases, with an average period of 10.6 days, in most cases (87 percent, 33/38) occurring in children less than 12 months of age. Although an association between this age group and the presentation of more severe clinical symptoms was observed, such as bronchiolitis in 51 percent (27/53) of the patients and pneumonia in 19 percent (10/53), no patients died. RSV was found from February to August, with the highest incidence in May. Conclusions: RSV is an important agent that causes ARIs; the clinical manifestations varied from mild to severe and patients frequently required hospitalization; RSV mostly affected children less than one year old.

Identification and molecular characterization of the infectious bursal disease virus (IBDV) from an outbreak in a broiler flock in midwestern Brazil

Para identificar e caracterizar o agente causador de um quadro clínico sugestivo de doença de Gumboro (DG) que afetou um plantel de frangos de corte com 34 dias de idade, em Buriti Alegre (estado de Goiás, centro-oeste do Brasil), no ano de 2001, procedeu-se uma combinação de métodos virológicos clássicos e modernos. Análises histopatológicas de bursas revelaram necrose, depleção de folículos linfóides, infiltração de heterófilos, edema e formação de cistos, lesões compatíveis com DG. A inoculação em ovos embrionados de galinhas SPF (specific pathogen-free) de uma suspensão de macerado de amostras de bursas resultou em mortalidade embrionária e lesões macroscópicas compatíveis com aquelas provocadas pelo VDIB. Amostras de bursas foram submetidas à técnica de transcrição reversa-reação em cadeia da polimerase (RT-PCR), utilizando-se oligonucleotídeos específicos para amplificação da região hipervariável do gene da VP2. Essa reação produziu um fragmento do tamanho esperado, que foi digerido pelas enzimas de restrição TaqI, StyI e SspI, mas não foi digerido com SacI. Este padrão foi o mesmo observado com cepas de VDIB hipervirulentas (vvVDIB). Análise da seqüência nucleotídica revelou a presença dos aminoácidos alanina, isoleucina e isoleucina nas posições 222, 256 e 294, respectivamente, característica dessas cepas. Além disso, análise filogenética realizada agrupou a cepa encontrada, denominada de BR-GO, com outras cepas de vvVDIB.

Polymerase chain reaction detection of adenovirus DNA sequences in human lymphocytes

Seqüências de DNA de adenovirus são freqüentemente encontradas em linfócitos humanos, tendo sido utilizadas para tal as técnicas de PCR e "Southern blot". Isto é possível apesar dessas células não serem permissivas à replicação de adenovírus, sugerindo persistência do genoma viral. Para investigar esse fenômeno, procuramos detectar em voluntários não sintomáticos DNA adenoviral e expressão do gene E1A. Amostras de DNA obtidas de glóbulos brancos periféricos de 51 voluntários, foram submetidas à PCR utilizando oligonucleotídeos para uma seqüência conservada do gene hexon, seguindo-se uma "nested PCR". Seqüências de adenovirus foram encontradas em 27 amostras (52,9 per center). Depois de mais de um ano, novas amostras desses voluntários positivos foram analisadas e em 70,8 per center dos casos o resultado foi mantido. Como isto poderia ser devido à persistência, decidimos verificar se o gene precoce E1A estava relacionado analisando sua expressão através de RT-PCR. Os resultados foram negativos para todas as amostras. O par de oligonucleotídeos desenvolvido para essa finalidade, que tem como alvo uma região conservada em E1A, permitiu detectar seqüências de adenovírus diretamente por PCR. A concordância encontrada entre essa análise e a pesquisa para o gene hexon foi de 84 per center. Nossos dados sugerem alta ocorrência e persistência de genoma adenoviral em linfócitos humanos, e indicam que uma região distinta de E1A é responsável pela persistência. Podemos também afirmar que a PCR para o gene E1A é uma boa opção quando se quer detectar adenovírus, evitando-se o alto risco de contaminação da "nested PCR" necessária para identificar a presença do gene hexon.