The importance of viral load in the severity of acute bronchiolitis in hospitalized infants.

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.

Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy.

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.

The importance of viral load in the severity of acute bronchiolitis in hospitalized infants

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.

Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR.

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 107 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

Silent circulation of dengue virus in Aedes albopictus (Diptera: Culicidae) resulting from natural vertical transmission.

Vertical transmission in Aedes aegypti and Aedes albopictus is considered a maintenance mechanism for dengue virus (DENV) during unfavorable conditions and may be implicated in dengue outbreaks. Since DENV infection dynamics vary among wild-type viruses and vector populations, vertical transmission rates can also vary between regions. However, even though São Paulo is the most populous city in the Americas and has experienced major dengue epidemics, natural vertical transmission had never been detected in this area before. Here we confirm and describe for the first time natural vertical transmission of DENV-3 in two pools of male Ae. albopictus from the city of São Paulo. The detection of DENV-3 in years when no human autochthonous cases of this serotype were recorded suggests that silent circulation of DENV-3 is occurring and indicates that green areas may be maintaining serotypes that are not circulating in the human population, possibly by a vertical transmission mechanism.

First report of two consecutive respiratory syncytial virus outbreaks by the novel genotypes ON-1 and NA-2 in a neonatal intensive care unit.

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 1,961g, 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.

Discovery of novel astrovirus and calicivirus identified in ruddy turnstones in Brazil.

Birds are the natural reservoir of viruses with zoonotic potential, as well as contributing to the evolution, emergence, and dissemination of novel viruses. In this study, we applied a high-throughput screening approach to identify the diversity of viruses in 118 samples of birds captured between October 2006 to October 2010 in the North and Northeast regions of Brazil. We found nearly complete genomes of novel species of astrovirus and calicivirus in cloacal swabs of ruddy turnstones (Arenaria interpres) collected in Coroa do Avião islet, Pernambuco State. These viruses are positive-sense single-stranded RNA with a genome of ~7 to 8 kb, and were designated as Ruddy turnstone astrovirus (RtAstV) and Ruddy turnstone calicivirus (RTCV), respectively. Phylogenetic analysis showed that RtAstV and RTCV grouped in a monophyletic clade with viruses identified from poultry samples (i.e., chicken, goose, and turkey), including viruses associated with acute nephritis in chickens. Attempts of viral propagation in monkey and chicken cell lines for both viruses were unsuccessful. Also, we found genomes related with viral families that infect invertebrates and plants, suggesting that they might be ingested in the birds' diet. In sum, these findings shed new light on the diversity of viruses in migratory birds with the notable characterization of a novel astrovirus and calicivirus.

Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil

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.

Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil.

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.

Novel avian paramyxovirus (APMV-15) isolated from a migratory bird in South America.

A novel avian paramyxovirus (APMV) isolated from a migratory bird cloacal swab obtained during active surveillance in April 2012 in the Lagoa do Peixe National Park, Rio Grande do Sul state, South of Brazil was biologically and genetically characterized. The nucleotide sequence of the full viral genome was completed using a next-generation sequencing approach. The genome was 14,952 nucleotides (nt) long, with six genes (3'-NP-P-M-F-HN-L-5') encoding 7 different proteins, typical of APMV. The fusion (F) protein gene of isolate RS-1177 contained 1,707 nucleotides in a single open reading frame encoding a protein of 569 amino acids. The F protein cleavage site contained two basic amino acids (VPKER↓L), typical of avirulent strains. Phylogenetic analysis of the whole genome indicated that the virus is related to APMV-10, -2 and -8, with 60.1% nucleotide sequence identity to the closest APMV-10 virus, 58.7% and 58.5% identity to the closest APMV-8 and APMV-2 genome, respectively, and less than 52% identity to representatives of the other APMVs groups. Such distances are comparable to the distances observed among other previously identified APMVs serotypes. These results suggest that unclassified/calidris_fuscicollis/Brazil/RS-1177/2012 is the prototype strain of a new APMV serotype, APMV-15.

Detection of hantavirus in bats from remaining rain forest in São Paulo, Brazil.

BACKGROUND: The significant biodiversity found in Brazil is a potential for the emergence of new zoonoses. Study in some places of the world suggest of the presence to hantavirus in tissues of bats. Researches of hantavirus in wildlife, out rodents, are very scarce in Brazil. Therefore we decided to investigate in tissues of different species of wild animals captured in the same region where rodents were detected positive for this virus. The present work analyzed ninety-one animals (64 rodents, 19 opossums, and 8 bats) from a region of the Atlantic forest in Biritiba Mirin City, São Paulo State, Brazil. Lungs and kidneys were used for RNA extraction. FINDINGS: The samples were screened for evidence of hantavirus infection by SYBR-Green-based real-time RT-PCR. Sixteen samples positive were encountered among the wild rodents, bats, and opossums. The detection of hantavirus in the lungs and kidneys of three marsupial species (Micoureus paraguayanus, Monodelphis ihering, and Didelphis aurita) as well in two species of bats (Diphylla ecaudata and Anoura caudifer) is of significance because these new hosts could represent an important virus reservoirs. CONCLUSIONS: The analysis of nucleotide sequences of the partial S segment revealed that these genes were more related to the Araraquara virus strains. This work reinforces the importance of studying hantavirus in different animal species and performing a continued surveillance before this virus spreads in new hosts and generated serious problems in public health.

Vigilância epidemiológica de vírus respiratórios humanos em amostras clínicas pela técnica de genescan-RT-PCR / GeneScan Reverse Transcription-PCR assay for surveillance of respiratory viruses in clinical samples of pediatric patients in Brazil

As doenças respiratórias agudas (DRAs) são as causas mais comuns de morbidez e mortalidade infantil mundial, podendo ser causadas por uma grande variedade de microorganismos. A fim de se detectar os vírus respiratórios mais comumente associados às infecções agudas do trato respiratório e traçar seu perfil epidemiológico, utilizamos um protocolo de GS RT-PCR (GeneScan Transcrição Reversa-Reação em Cadeia da Polimerase) para a rápida detecção simultânea do, vírus influenza A e B, parainfluenzavirus tipo 1, 2 e 3, picornavirus, metapneumovirus e o adenovírus. As amostras clínicas foram colhidas de crianças menores de cinco anos de idade, apresentando sintomas respiratórios, no Hospital Universitário (HU) da Universidade de São Paulo (USP), durante o ano de 2003. O GS RT-PCR se mostrou uma metodologia sensível e específica, capaz de detectar uma diversidade maior de agentes infecciosos do trato respiratório em relação à Imunofluorescência Indireta (IFI), reduzindo neste estudo a porcentagem de amostras negativas de 69,9 por cento (235 amostras) para 22 por cento (74 amostras).