Anti-respiratory syncytial virus and anti-herpes simplex virus activity of chemically engineered sulfated fucans from Cystoseira indica.

Seaweed polysaccharides, particularly sulfated ones, exhibited potent antiviral activity against a wide variety of enveloped viruses, such as herpes simplex virus and respiratory viruses. Different mechanisms of action were suggested, which may range from preventing infection to intracellular antiviral activity, at different stages of the viral cycle. Herein, we generated two chemically engineered sulfated fucans (C303 and C304) from Cystoseira indica by an amalgamated extraction-sulfation procedure using chlorosulfonic acid-pyridine/N,N-dimethylformamide and sulfur trioxide-pyridine/N,N-dimethylformamide reagents, respectively. These compounds exhibited activity against HSV-1 and RSV with 50 % inhibitory concentration values in the range of 0.75-2.5 µg/mL and low cytotoxicity at concentrations up to 500 µg/mL. The antiviral activities of chemically sulfated fucans (C303 and C304) were higher than the water (C301) and CaCl2 extracted (C302) polysaccharides. Compound C303 had a (1,3)-linked fucan backbone and was branched. Sulfates were present at positions C-2, C-4, and C-2,4 of Fucp, and C-6 of Galp residues of this polymer. Compound C304 had a comparable structure but with more sulfates at C-4 of Fucp residue. Both C303 and C304 were potent antiviral candidates, acting in a dose-dependent manner on the adsorption and other intracellular stages of HSV-1 and RSV replication, in vitro.

Zika virus infection impairs synaptogenesis, induces neuroinflammation, and could be an environmental risk factor for autism spectrum disorder outcome.

Zika virus (ZIKV) infection was first associated with Central Nervous System (CNS) infections in Brazil in 2015, correlated with an increased number of newborns with microcephaly, which ended up characterizing the Congenital Zika Syndrome (CZS). Here, we investigated the impact of ZIKV infection on the functionality of iPSC-derived astrocytes. Besides, we extrapolated our findings to a Brazilian cohort of 136 CZS children and validated our results using a mouse model. Interestingly, ZIKV infection in neuroprogenitor cells compromises cell migration and causes apoptosis but does not interfere in astrocyte generation. Moreover, infected astrocytes lost their ability to uptake glutamate while expressing more glutamate transporters and secreted higher levels of IL-6. Besides, infected astrocytes secreted factors that impaired neuronal synaptogenesis. Since these biological endophenotypes were already related to Autism Spectrum Disorder (ASD), we extrapolated these results to a cohort of children, now 6-7 years old, and found seven children with ASD diagnosis (5.14 %). Additionally, mice infected by ZIKV revealed autistic-like behaviors, with a significant increase of IL-6 mRNA levels in the brain. Considering these evidence, we inferred that ZIKV infection during pregnancy might lead to synaptogenesis impairment and neuroinflammation, which could increase the risk for ASD.

Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

SARS-CoV-2 Spike protein peptides displayed in the Pyrococcus furiosus RAD system preserve epitopes antigenicity, immunogenicity, and virus-neutralizing activity of antibodies.

Amongst the potential contribution of protein or peptide-display systems to study epitopes with relevant immunological features, the RAD display system stands out as a highly stable scaffold protein that allows the presentation of constrained target peptides. Here, we employed the RAD display system to present peptides derived from the SARS-CoV-2 Spike (S) protein as a tool to detect specific serum antibodies and to generate polyclonal antibodies capable of inhibiting SARS-CoV-2 infectivity in vitro. 44 linear S-derived peptides were genetically fused with the RAD scaffold (RAD-SCoV-epitopes) and screened for antigenicity with sera collected from COVID-19-infected patients. In a second step, selected RAD-SCoV-epitopes were used to immunize mice and generate antibodies. Phenotypic screening showed that some of these antibodies were able to recognize replicating viral particles in VERO CCL-81 and most notably seven of the RAD-SCoV-epitopes were able to induce antibodies that inhibited viral infection. Our findings highlight the RAD display system as an useful platform for the immunological characterization of peptides and a potentially valuable strategy for the design of antigens for peptide-based vaccines, for epitope-specific antibody mapping, and for the development of antibodies for diagnostic and therapeutic purposes.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Genetic diversity of adenovirus in neotropical bats from Brazil.

Bats can harbor a diversity of viruses, such as adenovirus. Ten different species of bat adenoviruses (BtAdV A to J) have been previous described worlwide. In Brazil, BtAdV was described in three species of phyllostomid species: Artibeus lituratus, Desmodus rotundus, and Sturnira lilium. There are around 180 bat species in Brazil, with 67% inhabiting the Atlantic Forest, with few information about the circulation of BtAdV in this biome. We aimed to describe the molecular detection and the phylogenetic characterization and suggest a classification of BtAdVs circulating in bats from the Brazilian Atlantic Forest. We collected 382 oral and rectal swabs from 208 bats between 2014-2015 and 2020-2021 from São Paulo, Pernambuco, and Santa Catarina Brazilian states. The adenovirus detection was done by a nested PCR targeting the DNA polymerase gene, and all positive samples were sequenced by the Sanger method. The phylogenetic analyses were based on the amino acid sequences using the MEGA 7 and BEAST software. We obtained 16 positive animals (detection rate 7.7%) belonging to seven bat species: Artibeus lituratus, Carollia perspicillata, Sturnira lilium, Molossus molossus, and the first record of Phyllostomus discolor, Eptesicus diminutus, and Myotis riparius. The phylogenetic analysis based on partial amino acid sequences showed that all obtained AdV sequences belong to the Mastadenovirus genus. We observed a high genetic diversity of BtAdV and identified eleven potential BtAdV species circulating in Brazil (BtAdV K to U). Our results contribute to the epidemiological surveillance of adenovirus, increasing the knowledge about the viral diversity and the distribution of AdV in bats from the Atlantic Forest.

Impact of an autophagy-inducing peptide on immunogenicity and protection efficacy of an adenovirus-vectored SARS-CoV-2 vaccine.

Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

First detection of Psittacid alphaherpesvirus 5 and coinfection with beak and feather disease virus in naturally infected captive ringneck parakeets (Psittacula krameri) in Brazil.

This study describes a case report in captive rose-ringed parakeets (Psittacula krameri) that developed clinical signs and eventually died after introducing new birds without quarantine. Bronchopneumonia and airsacculitis with syncytial cells associated with intranuclear inclusion bodies were found. Herpesvirus was detected in lungs and liver by PCR, and a nearly complete genome sequence of a Psittacid alphaherpesvirus 5 was obtained from the lung of a bird. Metagenomic analysis also identified beak and feather disease virus in the same samples. The study also highlights the importance of quarantine for avoiding the introduction of new diseases in captive aviaries.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Nano-multilamellar lipid vesicles loaded with a recombinant form of the chikungunya virus E2 protein improve the induction of virus-neutralizing antibodies.

Chikungunya virus (CHIKV) is responsible for a self-limited illness that can evolve into long-lasting painful joint inflammation. In this study, we report a novel experimental CHIKV vaccine formulation of lipid nanoparticles loaded with a recombinant protein derived from the E2 structural protein. This antigen fragment, designated ∆E2.1, maintained the antigenicity of the native viral protein and was specifically recognized by antibodies induced in CHIKV-infected patients. The antigen has been formulated into nanoparticles consisting of nano-multilamellar vesicles (NMVs) combined with the adjuvant monophosphoryl lipid A (MPLA). The vaccine formulation demonstrated a depot effect, leading to controlled antigen release, and induced strong antibody responses significantly higher than in mice immunized with the purified protein combined with the adjuvant. More relevantly, E2-specific antibodies raised in mice immunized with ∆E2.1-loaded NMV-MPLA neutralized CHIKV under in vitro conditions. Taken together, the results demonstrated that the new nanoparticle-based vaccine formulation represents a promising approach for the development of effective anti-CHIKV vaccines.

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.

Frequency of congenital cytomegalovirus infections in newborns in the Sao Paulo State, 2010-2018.

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.

Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil.

Reticuloendotheliosis viruses (REVs) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. Reticuloendotheliosis viruses are present worldwide and recently have been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report here the presence of REV in Brazil, detected in Muscovy Ducks (Cairina moschata), Wild Turkeys (Meleagris gallopavo), and chickens (Gallus gallus) at a relatively high prevalence (16.8%). Phylogenetic analysis indicated a close relationship of these strains to variants in the US. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

Natural versus recombinant viral antigens in SARS-COV-2 serology: challenges in optimizing laboratory diagnosis of COVID19

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.

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.

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool / Teste rápido de detecção de antígenos para o diagnóstico do Vírus Sincicial Respiratório como ferramenta de diagnóstico

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.

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.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

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.

Poor outcome of acute respiratory infection in young children with underlying health condition in Brazil.

OBJECTIVES: It is well established that respiratory viruses are an important cause of hospitalizations in young children worldwide, but data are limited on the contribution of specific viruses to severe illness in South America. We describe clinical and laboratory findings from prospective surveillance for acute respiratory infections at a tertiary hospital in São Paulo, Brazil. METHODS: We screened children < 2 years old with acute respiratory tract infections admitted to an urban tertiary hospital for respiratory viruses from March 2008 through February 2010, using polymerase chain reaction assays. RESULTS: Respiratory viruses were identified in 378 (53%) of the 715 samples analyzed. Respiratory syncytial virus was the most commonly identified virus (52%), followed by adenovirus (27%) and Human metapneumovirus (12%). More than one virus was identified in 19% of specimens. Almost half of the samples (46%) were from children with underlying health conditions. We demonstrated that compared to the previously healthy group, those with comorbidities had a worse outcome in terms of severity, with prolonged hospital stay and more need of intensive care. CONCLUSION: Identification of this high-risk population along with strategies for fast diagnosis might each help to reduce morbidity and mortality in this group.