OBJECTIVE: The relationship between viral load and the clinical evolution of bronchiolitis is controversial. Therefore, we aimed to analyze viral loads in infants hospitalized for bronchiolitis. METHODS: We tested for the presence of human respiratory syncytial virus (HRSV) or human rhinovirus (HRV) using quantitative molecular tests of nasopharyngeal secretions and recorded severity outcomes. RESULTS: We included 70 infants [49 (70%) HRSV, 9 (13%) HRV and 12 (17%) HRSV+HRV]. There were no differences among the groups according to the outcomes analyzed individually. Clinical scores showed greater severity in the isolated HRSV infection group. A higher isolated HRSV viral load was associated with more prolonged ventilatory support, oxygen therapy, and hospitalization days, even after adjustment for the age and period of nasopharyngeal secretion collection. In the co-infection groups, there was a longer duration of oxygen therapy when the HRSV viral load was predominant. Isolated HRV infection and co-infection with a predominance of HRV were not associated with severity. CONCLUSION: Higher HRSV viral load in isolated infections and the predominance of HRSV in co-infections, independent of viral load, were associated with greater severity. These results contribute to the development of therapeutic and prophylactic approaches and a greater understanding of the pathophysiology of bronchiolitis.

Resumo Objetivo O vírus sincicial respiratório é um patógeno frequentemente envolvido em surtos nosocomiais. Embora vários estudos tenham relatado tais surtos em unidades de terapia intensiva neonatal, os dados epidemiológicos moleculares são escassos. Neste artigo, descrevemos dois surtos consecutivos de vírus sincicial respiratório causados pelos genótipos ON-1 e NA-2 em uma unidade de terapia intensiva neonatal em São Paulo, Brasil. Métodos Uma busca prospectiva por vírus sincicial respiratório foi realizada após o diagnóstico do caso índice e outros quatro recém-nascidos sintomáticos na unidade de terapia intensiva neonatal. Amostras de aspirado nasofaríngeo de todos os pacientes da unidade de terapia intensiva neonatal foram testadas para 17 vírus respiratórios com reação em cadeia da polimerase via transcriptase reversa em tempo real. A genotipagem realizada utilizando sequenciamento de nucleotídeos. Resultados De maio a agosto de 2013, foram detectados dois surtos diferentes na unidade de terapia intensiva neonatal. Vinte e quatro crianças foram infectadas com vírus sincicial respiratório-A (10 e 14 com os genótipos ON-1 e NA-2, respectivamente). A média da idade dos lactentes era de 10 dias, o peso médio ao nascer foi de 1961 g e a idade gestacional média de 33 semanas. Fatores de risco (doença cardíaca, doença pulmonar e prematuridade) estavam presentes em 80% e 85,7% dos bebês nos grupos ON-1 e NA-2, respectivamente. No total, 45,8% dos lactentes eram assintomáticos e 20,8% necessitaram de ventilação mecânica. Não foram detectadas coinfecções durante os surtos. Conclusões Bebês em unidade de terapia intensiva neonatal que desenvolvem sintomas respiratórios abruptos devem ser testados para vírus respiratórios, especialmente o vírus sincicial respiratório. Mesmo na ausência de sintomas graves, a detecção de vírus sincicial respiratório pode prevenir a transmissão nosocomial através de medidas de controle de infecção. Um melhor entendimento da epidemiologia molecular do vírus sincicial respiratório é essencial para o desenvolvimento de novas vacinas e drogas antivirais contra o vírus sincicial respiratório.

Abstract Objective: Respiratory syncytial virus is a pathogen frequently involved in nosocomial outbreaks. Although several studies have reported nosocomial outbreaks in neonatal intensive care units, molecular epidemiology data are scarce. Here, the authors describe two consecutive respiratory syncytial virus outbreaks caused by genotypes ON-1 and NA-2 in a neonatal intensive care unit in São Paulo, Brazil. Methods: A prospective search for respiratory syncytial virus was performed after diagnosing the index case and four other symptomatic newborns in the neonatal intensive care unit. Nasopharyngeal aspirate samples of all patients in the neonatal intensive care unit were tested for 17 respiratory viruses using real-time reverse transcriptase polymerase chain reaction. Genotyping was performed using nucleotide sequencing. Results: From May to August 2013, two different outbreaks were detected in the neonatal intensive care unit. A total of 20 infants were infected with respiratory syncytial virus-A (ten and 14 with ON-1 and NA-2 genotypes, respectively). The mean age of the infants was 10 days, mean birth weight was 1,961 g, and the mean gestational age was 33 weeks. Risk factors (heart disease, lung disease, and prematurity) were present in 80% and 85.7% of infants in the ON-1 and NA-2 groups, respectively. In total, 45.8% of infants were asymptomatic and 20.8% required mechanical ventilation. Coinfections were not detected during the outbreaks. Conclusions: Infants in a neonatal intensive care unit who develop abrupt respiratory symptoms should be tested for respiratory viruses, especially respiratory syncytial virus. Even in the absence of severe symptoms, respiratory syncytial virus detection can prevent nosocomial transmission through infection control measures. A better understanding of respiratory syncytial virus molecular epidemiology is essential for developing new vaccines and antiviral drugs against respiratory syncytial virus.

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

ABSTRACT Human cytomegalovirus (HCMV) infections remain a neglected public health issue. The aim of the present study was to evaluate the frequency of HCMV congenital infections in newborns up to 1 month in the Sao Paulo State, from 2010 to 2018. The molecular characterization of HCMV-positive samples was also undertaken. Urine samples from 275 potential congenital HCMV-infected patients were tested by real-time Polymerase Chain Reaction (qPCR). HCMV-positive samples were amplified by conventional PCR targeting the UL89 gene, sequenced and searched for mutations. A total of 32 (11.6%) positive-HCMV cases were detected (mean Ct 30.59); mean and median age of 10.3 and 6 days old, respectively. Children aged between 0-3 weeks had higher HCMV detection rates (84.4%; 27/32). UL89 gene was successfully sequenced in two samples, both classified as the human betaherpesvirus 5. No described resistance-associated mutations were identified. A routine screening in newborns coupled with the genetic characterization of key viral genes is vital to decrease sequels associated with congenital HCMV infections.

Resumo Objetivo: Caracterizar o papel do VPH-4 e suas características clínicas em crianças brasileiras com menos de dois anos de idade com infecções agudas do trato respiratório inferior. Métodos: Ensaios em tempo real foram utilizados para identificar tipos de VPH e outros vírus respiratórios comuns em aspirados nasofaríngeos. Mil e duas crianças com doença aguda do trato respiratório inferior foram inscritas para participar de fevereiro de 2008 a agosto de 2010. Resultados: 104 (10,4%) pacientes eram VPH positivos, dos quais 60 (57,7%) eram positivos para VPH-3, 30 (28,8%) para VPH-4, 12 (11,5%) para VPH-1 e dois (1,9%) para VPH-2. Sete (6,7%) pacientes apresentaram mais de um tipo de VPH detectado. Os sintomas mais frequentes foram tosse e taquipneia, semelhantes a outras infecções respiratórias virais. As manifestações clínicas não diferiram de forma significativa entre as infecções por VPH-1, -2, -3 e -4. Os VPH-1, -3 e -4 estavam presentes na população estudada ao longo dos três anos de vigilância, e o VPH-3 foi o tipo predominante identificado nos primeiros dois anos. Conclusão: Os VPHs contribuem substancialmente para a DRA pediátrica no Brasil com quase 30% dessa contribuição atribuível ao VPH-4.

Abstract Objective: Characterize the role of human parainfluenza virus and its clinical features in Brazilian children under 2 years of age presenting with acute lower respiratory tract infections. Methods: Real-time assays were used to identify strains of human parainfluenza virus and other common respiratory viruses in nasopharyngeal aspirates. One thousand and two children presenting with acute lower respiratory tract illnesses were enrolled from February 2008 to August 2010. Results: One hundred and four (10.4%) patients were human parainfluenza virus positive, of whom 60 (57.7%) were positive for human parainfluenza virus-3, 30 (28.8%) for human parainfluenza virus-4, 12 (11.5%) for human parainfluenza virus-1, and two (1.9%) for human parainfluenza virus-2. Seven (6.7%) patients had more than one strain of human parainfluenza virus detected. The most frequent symptoms were tachypnea and cough, similar to other viral respiratory infections. Clinical manifestations did not differ significantly between human parainfluenza virus-1, -2, -3, and -4 infections. Human parainfluenza virus-1, -3, and -4 were present in the population studied throughout the three years of surveillance, with human parainfluenza virus-3 being the predominant type identified in the first two years. Conclusion: Human parainfluenza viruses contribute substantially to pediatric acute respiratory illness (ARI) in Brazil, with nearly 30% of this contribution attributable to human parainfluenza virus-4.

ABSTRACT The red-tailed Amazon parrot (Amazona brasiliensis) is a threatened species of psittacine bird that inhabit coastal regions of Brazil. In view of the threat of this species, the aim of this study was to perform a health evaluation in wild nestlings in Rasa Island, determining the prevalence of enterobacteria and infectious agents according to type of nest. Blood samples were collected from 64 birds and evaluated for antibodies of Chlamydia psittaci by commercial dot-blot ELISA. Cloacal and oropharyngeal swabs samples were collected from 23 birds from artificial wooden nests, 15 birds from PVC nests and 2 birds from natural nests for microbiological analysis. Swab samples were collected from 58 parrots for C. psittaci detection by PCR and from 50 nestlings for Avian Influenza, Newcastle Disease and West Nile viruses’ detection analysis by real-time RT-PCR. Ten bacterial genera and 17 species were identified, and the most prevalent were Escherichia coli and Klebsiella oxytoca. There was no influence of the type of nest in the nestlings’ microbiota. All samples tested by ELISA and PCR were negative. There is currently insufficient information available about the health of A. brasiliensis and data of this study provide a reference point for future evaluations and aid in conservation plans.

Abstract INTRODUCTION: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

Resumo Objetivo: Avaliar o teste QuickVue® RSV Test Kit (QUIDEL Corp, CA, EUA) para o diagnóstico rápido do vírus sincicial respiratório em crianças com doença respiratória aguda, comparandoo com a imunofluorescência indireta como padrão ouro. Visto que, no Brasil, testes rápidos para detecção de antígenos para vírus sincicial respiratório não são rotineiramente utilizados como ferramenta de diagnóstico, exceto para Dengue e Influenza. Métodos: Um total de 486 amostras de aspirado de nasofaringe de crianças menores de 5 anos com doença respiratória aguda, coletadas entre dezembro de 2013 e agosto de 2014, foram analisadas por imunofluorescência e pelo teste QuickVue®. Amostras com resultados discordantes entre os métodos foram submetidas a PCR em tempo real e sequenciamento. Resultados: Das 313 amostras positivas por IFI, 282 foram positivas no teste rápido (90%), 2 amostras foram positivas apenas no teste rápido (0.6%), 33 apenas na imunofluorescência (10.5%) e 171 foram negativas em ambos os métodos. As 35 amostras com resultados discordantes foram testadas por PCR em tempo real, sendo que duas que foram positivas apenas no teste rápido e 5 apenas na imunofluorescência confirmaram-se positivas. Não houve relação entre a ausência de positividade no teste QuickVue® com a carga ou com a cepa viral. O teste QuickVue® mostrou sensibilidade de 90.1%, especificidade 98.9%, valor preditivo positivo 99.3%, valor preditivo negativo de 94.6%, acurácia de 93.2% e índice de concordância de 0.85 em comparação à imunofluorescência. Conclusões: Nosso estudo demonstrou que o teste QuickVue® RSV pode ser efetivo na detecção precoce do vírus sincicial respiratório em amostras de aspirado de nasofaringe e é confiável como uma ferramenta de diagnósticos em pediatria.

Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.

Objetivo: a rubéola, durante os primeiros estágios da gravidez, pode levar a graves defeitos congênitos, conhecidos como síndrome da rubéola congênita (SRC). Amostras de gestantes com sintomas e suspeitas da rubéola congênita foram coletadas entre 1996 e 2008. Métodos: um total de 23 amostras de fluido amniótico, 16 amostras de sangue fetal, um produto da concepção e uma placenta foram analisados por sorologia e PCR. Resultados: todas as gestantes apresentaram sorologia positiva para IgG/IgM para o vírus da rubéola. Entre os recém-nascidos, 14 apresentaram anticorpos IgG positivos e 11 foram os anticorpos IgM positivos. Das 25 amostras analisadas neste estudo, 24 eram positivas por RT-PCR. Alterações na ultrassonografia foram encontradas em 15 (60%) dos 25 fetos infectados com o vírus da rubéola. Morte fetal e aborto espontâneo foram reportados em 10 (40%) dos 25 casos analisados. O vírus da rubéola foi amplificado por PCR em todos os fetos que apresentaram alterações na ultrassonografia, compatíveis com a rubéola. Morte fetal e aborto foram relatados em 10 dos 25 casos analisados. Conclusão: os resultados mostraram que os ensaios moleculares são ferramentas importantes para o diagnóstico precoce da rubéola e da síndrome da rubéola congênita.

Objective: rubella during the early stages of pregnancy can lead to severe birth defects known as congenital rubella syndrome (CRS). Samples collected from pregnant women with symptoms and suspected of congenital rubella infection between 1996 and 2008 were analyzed. Methods: a total of 23 amniotic fluid samples, 16 fetal blood samples, 1 product of conception and 1 placenta were analyzed by serology and RT-PCR. Results: all patients presented positive serology for IgG / IgM antibodies to rubella virus. Among neonates, 16 were IgG-positive, 9 were IgM-positive and 4 were negative for both antibodies. Of the 25 samples analyzed in this study, 24 were positive by RT-PCR. Changes in ultrasound were found in 15 (60%) of 25 fetuses infected with rubella virus. Fetal death and miscarriage were reported in 10 (40%) of the 25 cases analyzed. The rubella virus was amplified by PCR in all fetuses with abnormal ultrasound compatible with rubella. Fetal death and abortion were reported in 10 of 25 cases analyzed. Conclusion: this study, based on primary maternal rubella infection definitely confirms the good sensitivity and specificity of RT-PCR using amniotic fluid and ultrasound. The results showed that molecular assays are important tools in the early diagnosis of rubella and congenital rubella syndrome.

OBJETIVO: Caracterizar e comparar aspectos clínicos, epidemiológicos e laboratoriais delactentes com evidências de infecção aguda do trato respiratório inferior (IATRI) associada à detecção do adenovírus (ADV) ou do vírus sincicial respiratório (VSR). MÉTODOS: Um estudo preliminar de vigilância de infecções respiratórias desenvolveu coleta de aspirado nasofaríngeo (ANF) para pesquisa viral, vinculada ao preenchimento de protocolo padrão, de menores de dois anos internados com quadro de IATRI em hospital universitário, entre março de 2008 e agosto de 2011. Utilizou-se técnica da reação em cadeia da polimerase (PCR) para oito vírus: ADV, VSR, metapneumovírus, parainfluenza 1, 2 e 3 e influenza A e B. Foram selecionados para comparações os casos com ANF coletado nas primeiras 24 horas da admissão, resultado de hemocultura negativo e detecção exclusiva de ADV (grupo Gadv) ou VSR (grupo Gvsr). RESULTADOS: O estudo preliminar incluiu coleta de 1.121 amostras de ANF, sendo 813 coletadas nas primeiras 24 h da admissão, das quais 50,3% foram positivas para ao menos um dos vírus, com VSR em primeiro lugar, em 27,3%, e ADV em segundo, em 15,8% dos casos pesquisados. Dentre os aspectos analisados nos grupos Gadv (n = 58) e Gvsr (n = 134), destacaram-se a média da idade mais elevada, maior frequência da prescrição de antibióticos e medianas mais elevadas para contagem total de leucócitos e valores da proteína C-reativa no Gadv. CONCLUSÕES: A PCR utilizada pode detectar formas persistentes/latentes de ADV, aspecto aser considerado ao interpretar os resultados. Estudos complementares com técnicas diagnósticas quantitativas, por exemplo, poderiam evidenciar a importância da elevada frequência verificada.

OBJECTIVE: To characterize and compare clinical, epidemiological, and laboratory aspects ofinfants with acute lower respiratory infection (ALRI) associated with the detection of adenovirus(ADV) or respiratory syncytial virus (RSV). METHODS: A preliminary respiratory infection surveillance study collected samples of nasopharyngeal aspirate (NPA) for viral research, linked to the completion of a standard protocol, from children younger than two years admitted to a university hospital with ALRI, between March of 2008 and August of 2011. Polymerase chain reaction (PCR) was used for eight viruses: ADV, RSV, metapneumovirus, Parainfluenza 1, 2, and 3, and Influenza A and B. Cases with NPA collectedduring the first 24 hours of admission, negative results of blood culture, and exclusive detection of ADV (Gadv group) or RSV (Grsv group) were selected for comparisons. RESULTS: The preliminary study included collection of 1,121 samples of NPA, 813 collected in thefirst 24 hours of admission, of which 50.3% were positive for at least one virus; RSV was identifiedin 27.3% of cases surveyed, and ADV was identified in 15.8%. Among the aspects analyzed inthe Gadv (n = 58) and Grsv (n = 134) groups, the following are noteworthy: the higher meanage, more frequent prescription of antibiotics, and the highest median of total white blood cellcount and C-reactive protein values in Gadv. CONCLUSIONS: PCR can detect persistent/latent forms of ADV, an aspect to be considered wheninterpreting results. Additional studies with quantitative diagnostic techniques could elucidatethe importance of the high frequency observed.

Introduction This study assessed the viability of the rabies virus in the argasid tick Carios fonsecai following experimental infection. Methods The mouse inoculation test (MIT), fluorescent antibody test (FAT) and polymerase chain reaction (PCR) were used. The rabies virus was administered to ticks via the intra-coelomic route, and the ticks were sacrificed at different time points. Results The inoculated ticks were negative for rabies according to the MIT. Ticks macerated with rabies virus were positive according to the MIT and FAT. All of the tick lots tested by PCR were positive. Conclusions The rabies virus became unviable shortly after its inoculation into tick bodies. Ticks are not likely to play an important role in the epidemiology of rabies.

Viruses are the major cause of lower respiratory tract infections in childhood and the main viruses involved are Human Respiratory Syncytial Virus (HRSV), Human Metapneumovirus (HMPV), Influenzavirus A and B (FLUA and FLUB), Human Parainfluenza Virus 1, 2 and 3 (HPIV1, 2 and 3) and Human Rhinovirus (HRV). The purposes of this study were to detect respiratory viruses in hospitalized children younger than six years and identify the influence of temperature and relative air humidity on the detected viruses. Samples of nasopharyngeal washes were collected from hospitalized children between May/2004 and September/2005. Methods of viral detection were RT-PCR, PCR and HRV amplicons were confirmed by hybridization. Results showed 54% (148/272) of viral positivity. HRSV was detected in 29% (79/272) of the samples; HRV in 23.1% (63/272); HPIV3 in 5.1% (14/272); HMPV in 3.3% (9/272); HPIV1 in 2.9% (8/272); FLUB in 1.4% (4/272), FLUA in 1.1% (3/272), and HPIV2 in 0.3% (1/272). The highest detection rates occurred mainly in the spring 2004 and in the autumn 2005. It was observed that viral respiratory infections tend to increase as the relative air humidity decreases, showing significant association with monthly averages of minimal temperature and minimal relative air humidity. In conclusion, viral respiratory infections vary according to temperature and relative air humidity and viral respiratory infections present major incidences it coldest and driest periods.