Rational design of large flat nitrogen-doped graphene oxide quantum dots with green-luminescence suitable for biomedical applications.

Rational synthesis and simple methodology for the purification of large (35-45 nm in lateral size) and flat (1.0-1.5 nm of height) nitrogen-doped graphene oxide quantum dots (GOQDs) are presented. The methodology allows robust metal-free and acid-free preparation of N-GOQDs with a yield of about 100% and includes hydrothermal treatment of graphene oxide with hydrogen peroxide and ammonia. It was demonstrated that macroscopic impurities can be separated from N-GOQD suspension by their coagulation with 0.9% NaCl solution. Redispersible in water and saline solutions, particles of N-GOQDs were characterized using tip-enhanced Raman spectroscopy (TERS), photoluminescent, XPS, and UV-VIS spectroscopies. The size and morphology of N-GOQDs were studied by dynamic light scattering, AFM, SEM, and TEM. The procedure proposed allows nitrogen-doped GOQDs to be obtained, having 60-51% of carbon, 34-45% of oxygen, and up to 7.2% of nitrogen. The N-GOQD particles obtained in two hours of synthesis contain only pyrrolic defects of the graphene core. The fraction of pyridine moieties grows with the time of synthesis, while the fraction of quaternary nitrogen declines. Application of TERS allows demonstration that the N-GOQDs consist of a graphene core with an average crystallite size of 9 nm and an average distance between nearest defects smaller than 3 nm. The cytotoxicity tests reveal high viability of the monkey epithelial kidney cells Vero in the presence of N-GOQDs in a concentration below 60 mg L-1. The N-GOQDs demonstrate green luminescence with an emission maximum at 505 nm and sedimentation stability in the cell culture medium.

COVID-19 diagnosis by RT-qPCR in alternative specimens.

BACKGROUND: The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES: The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS: We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS: When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS: Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

Inactivation of avian influenza viruses by hydrostatic pressure as a potential vaccine development approach.

Vaccines are a recommended strategy for controlling influenza A infections in humans and animals. Here, we describe the effects of hydrostatic pressure on the structure, morphology and functional characteristics of avian influenza A H3N8 virus. The effect of hydrostatic pressure for 3 h on H3N8 virus revealed that the particles were resistant to this condition, and the virus displayed only a discrete conformational change. We found that pressure of 3 kbar applied for 6 h was able to inhibit haemagglutination and infectivity while virus replication was no longer observed, suggesting that full virus inactivation occurred at this point. However, the neuraminidase activity was not affected at this approach suggesting the maintenance of neutralizing antibody epitopes in this key antigen. Our data bring important information for the area of structural virology of enveloped particles and support the idea of applying pressure-induced inactivation as a tool for vaccine production.

COVID-19 diagnosis by RT-qPCR in alternative specimens

BACKGROUND The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

Low lineage diversity and increased virulence of group C Streptococcus dysgalactiae subsp. equisimilis.

Introduction. In some species, the population structure of pathogenic bacteria is clonal. However, the mechanisms that determine the predominance and persistence of specific bacterial lineages of group C Streptococcus remain poorly understood. In Brazil, a previous study revealed the predominance of two main lineages of Streptococcus dysgalactiae subsp. equisimilis (SDSE).Aim. The aim of this study was to assess the virulence and fitness advantages that might explain the predominance of these SDSE lineages for a long period of time.Methodology. emm typing was determined by DNA sequencing. Adhesion and invasion tests were performed using human bronchial epithelial cells (16HBE14o-). Biofilm formation was tested on glass surfaces and the presence of virulence genes was assessed by PCR. Additionally, virulence was studied using Caenorhabditis elegans models and competitive fitness was analysed in murine models.Results. The predominant lineages A and B were mostly typed as emm stC839 and stC6979, respectively. Notably, these lineages exhibited a superior ability to adhere and invade airway cells. Furthermore, the dominant lineages were more prone to induce aversive olfactory learning and more likely to kill C. elegans. In the competitive fitness assays, they also showed increased adaptability. Consistent with the increased virulence observed in the ex vivo and in vivo models, the predominant lineages A and B showed a higher number of virulence-associated genes and a superior ability to accumulate biofilm.Conclusion. These results suggest strongly that this predominance did not occur randomly but rather was due to adaptive mechanisms that culminated in increased colonization and other bacterial properties that might confer increased bacteria-host adaptability to cause disease.

Stability of different influenza subtypes: How can high hydrostatic pressure be a useful tool for vaccine development?

BACKGROUND: Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation. METHODS: Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes. RESULTS: HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses. CONCLUSIONS: HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity.

Homeopathic treatments modify inflammation but not behavioral response to influenza antigen challenge in BALB/c mice.

BACKGROUND: Influenza affects thousands of people worldwide every year, motivating the development of new therapies. In this work, the effects of two homeopathic preparations (influenza biotherapies and thymulin) were chosen following two different rationales: isotherapy and endo-isotherapy models. The homeopathic effects were evaluated individually considering the inflammatory and behavioral responses against influenza virus antigen were studied in BALB/c mice. METHODS: Male adult mice were treated orally and blindly for 21 days with highly diluted influenza virus or with thymulin, and were divided in two sets of experiments. The first series of experiments aimed to describe their behavior, using an open field (OF) device. In the second series, mice were challenged subcutaneously with influenza hemagglutinin antigen (7 µg/200 µl) at day 21. At day 42, behavior and inflammation response were evaluated. RESULTS: No behavioral changes were seen in OF tests at any time point after treatments. Flow cytometry and morphometry revealed significant changes in T and B cell balance after influenza antigen challenge, varying according to treatment. CONCLUSION: The results show that both homeopathic treatments induced subtle changes in acquired immune anti-viral response regulation. A deeper understanding of the mechanism could elucidate their possible use in influenza epidemiological situations.

Homeopathic medicines for prevention of influenza and acute respiratory tract infections in children: blind, randomized, placebo-controlled clinical trial.

BACKGROUND: Influenza and its complications are common at all ages, especially in children. Vaccines and anti-influenza drugs aim to prevent it. Preventative approaches with favorable risk profiles should be considered for flu, particularly since the evidence of the efficacy of anti-viral drugs is debated. METHODS: This pragmatic clinical trial was conducted in the Brazilian Public Health System in Petrópolis (BPHSP) with children aged from 1 to 5 years old. The medications used were mainly selected based on in vitro experiments (InfluBio), and in successful qualitative clinical experiences (Homeopathic Complex). Following informed parental consent, subjects were randomly distributed, in a blind manner, to three experimental groups: Homeopathic Complex, Placebo, and InfluBio. BPHSP health agents collected flu and acute respiratory infection symptomatic episodes monthly following the established protocol. The number of these episodes was registered in one year (2009-2010). RESULTS: Out of the 600 children recruited, 445 (74.17%) completed the study (149: Homeopathic complex; 151: Placebo; 145: InfluBio). The number of flu and acute respiratory infection symptomatic episodes detected in this clinical trial was low; however, it was different between homeopathic groups and placebo (p < 0.001). In the first year post-intervention, 46/151 (30.5%) of children in the placebo group developed 3 or more flu and acute respiratory infection episodes, while there was no episode in the group of 149 children who used Homeopathic Complex, and only 1 episode in the group of 145 (1%) children who received InfluBio. CONCLUSION: These results suggested that the use of homeopathic medicines minimized the number of flu and acute respiratory infection symptomatic episodes in children, signalizing that the homeopathic prophylactic potential should be investigated in further studies.

Intranasal Immunization with Pressure Inactivated Avian Influenza Elicits Cellular and Humoral Responses in Mice.

Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.

Frequency of viral etiology in symptomatic adult upper respiratory tract infections

Aims: To determine the frequency of viral pathogens causing upper respiratory tract infections in non-hospitalized, symptomatic adults in the city of Rio de Janeiro. Methods: Respiratory samples (nasal/throat swabs) were collected between August 2010 and November 2012 and real time PCR was used to detect different viral pathogens. Results: Viruses were detected in 32.1% (43/134) of samples from 101 patients. Specifically, 9% (12/134) were positive for HBoV, 8.2% (11/134) were positive for HAdV, 5.2% (7/134) were positive for HRV, and 1.5% (2/134) were positive for FLUBV or HMPV, as single infections. HRSV-A, HPIV-3, and HCoV-HKU1 were detected in one (0.75%) sample each. Co-infections were detected in 4.8% (6/134) of the samples. Peaks of viral infections were observed in March, April, May, August, and October. However, positive samples were detected all year round. Only 23.3% (10/43) of the positive samples were collected from patients with febrile illness. Conclusion: Results presented in this report suggest that respiratory viral infections are largely under diagnosed in immunocompetent adults. Although the majority of young adult infections are not life-threatening they may impose a significant burden, especially in developing countries since these individuals represent a large fraction of the working force. .

Frequency of viral etiology in symptomatic adult upper respiratory tract infections.

AIMS: To determine the frequency of viral pathogens causing upper respiratory tract infections in non-hospitalized, symptomatic adults in the city of Rio de Janeiro. METHODS: Respiratory samples (nasal/throat swabs) were collected between August 2010 and November 2012 and real time PCR was used to detect different viral pathogens. RESULTS: Viruses were detected in 32.1% (43/134) of samples from 101 patients. Specifically, 9% (12/134) were positive for HBoV, 8.2% (11/134) were positive for HAdV, 5.2% (7/134) were positive for HRV, and 1.5% (2/134) were positive for FLUBV or HMPV, as single infections. HRSV-A, HPIV-3, and HCoV-HKU1 were detected in one (0.75%) sample each. Co-infections were detected in 4.8% (6/134) of the samples. Peaks of viral infections were observed in March, April, May, August, and October. However, positive samples were detected all year round. Only 23.3% (10/43) of the positive samples were collected from patients with febrile illness. CONCLUSION: Results presented in this report suggest that respiratory viral infections are largely under diagnosed in immunocompetent adults. Although the majority of young adult infections are not life-threatening they may impose a significant burden, especially in developing countries since these individuals represent a large fraction of the working force.

Full inactivation of human influenza virus by high hydrostatic pressure preserves virus structure and membrane fusion while conferring protection to mice against infection.

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.

Influenza A virus infection of healthy piglets in an abattoir in Brazil: animal-human interface and risk for interspecies transmission.

Asymptomatic influenza virus infections in pigs are frequent and the lack of measures for controlling viral spread facilitates the circulation of different virus strains between pigs. The goal of this study was to demonstrate the circulation of influenza A virus strains among asymptomatic piglets in an abattoir in Brazil and discuss the potential public health impacts. Tracheal samples (n = 330) were collected from asymptomatic animals by a veterinarian that also performed visual lung tissue examinations. No slaughtered animals presented with any noticeable macroscopic signs of influenza infection following examination of lung tissues. Samples were then analysed by reverse transcription-polymerase chain reaction that resulted in the identification of 30 (9%) influenza A positive samples. The presence of asymptomatic pig infections suggested that these animals could facilitate virus dissemination and act as a source of infection for the herd, thereby enabling the emergence of influenza outbreaks associated with significant economic losses. Furthermore, the continuous exposure of the farm and abattoir workers to the virus increases the risk for interspecies transmission. Monitoring measures of swine influenza virus infections and vaccination and monitoring of employees for influenza infection should also be considered. In addition regulatory agencies should consider the public health ramifications regarding the potential zoonotic viral transmission between humans and pigs.

Influenza A virus infection of healthy piglets in an abattoir in Brazil: animal-human interface and risk for interspecies transmission

Asymptomatic influenza virus infections in pigs are frequent and the lack of measures for controlling viral spread facilitates the circulation of different virus strains between pigs. The goal of this study was to demonstrate the circulation of influenza A virus strains among asymptomatic piglets in an abattoir in Brazil and discuss the potential public health impacts. Tracheal samples (n = 330) were collected from asymptomatic animals by a veterinarian that also performed visual lung tissue examinations. No slaughtered animals presented with any noticeable macroscopic signs of influenza infection following examination of lung tissues. Samples were then analysed by reverse transcription-polymerase chain reaction that resulted in the identification of 30 (9%) influenza A positive samples. The presence of asymptomatic pig infections suggested that these animals could facilitate virus dissemination and act as a source of infection for the herd, thereby enabling the emergence of influenza outbreaks associated with significant economic losses. Furthermore, the continuous exposure of the farm and abattoir workers to the virus increases the risk for interspecies transmission. Monitoring measures of swine influenza virus infections and vaccination and monitoring of employees for influenza infection should also be considered. In addition regulatory agencies should consider the public health ramifications regarding the potential zoonotic viral transmission between humans and pigs.

Impact of agr dysfunction on virulence profiles and infections associated with a novel methicillin-resistant Staphylococcus aureus (MRSA) variant of the lineage ST1-SCCmec IV.

BACKGROUND: A novel variant of the ST1-SCCmecIV methicillin-resistant Staphylococcus aureus (MRSA) lineage, mostly associated with nosocomial bloodstream infections (BSI), has emerged in Rio de Janeiro. Bacterial biofilm has been considered a major virulence factor in central venous catheter-associated BSI. The mechanisms involved in biofilm formation/accumulation are multifactorial and complex. Studies have suggested that biofilm production was affected in vitro and vivo for agr-null mutants of S. aureus. RESULTS: The impact of naturally occurring inhibition of agr signaling on virulence profiles and infections associated with the ST1 variant was investigated. agr dysfunction was detected in a significant percentage (13%) of the isolates with concomitant increase in biofilm accumulation in vitro and in vivo, and enhanced ability to adhere to and invade airway cells. The biofilm formed by these ST1 isolates was ica-independent and proteinaceous in nature. In fact, the improved colonization properties were paralleled by an increased expression of the biofilm-associated genes fnbA, spa and sasG. The transcription of sarA, a positive regulator of agr, was two-times reduced for the agr-dysfunctional MRSA. Remarkably, the agr inhibition was genetically stable. Indeed, agr-dysfunctional isolates succeed to colonize and cause both acute and chronic infections in hospitalized patients, and also to effectively accumulate biofilm in a mouse subcutaneous catheter implant model. CONCLUSION: The ability of agr-dysfunctional isolates to cause infections in humans and to form biofilm in the animal model suggests that therapeutic approaches based on agr-inactivation strategies are unlikely to be effective in controlling human-device infections caused by ST1 isolates. The increased biofilm accumulation associated with the acquisition of multiple antimicrobial resistant traits might have influenced (at least in part) the expansion of this USA400 related clone in our hospitals.

H3N2 homeopathic influenza virus solution modifies cellular and biochemical aspects of MDCK and J774G8 cell lines.

BACKGROUND: Influenza viruses cause highly contagious acute respiratory illnesses with significant mortality, especially among young children, elderly people, and individuals with serious medical conditions. This encourages the development of new treatments for human flu. Biotherapies are diluted solutions prepared from biological products compounded following homeopathic procedures. OBJECTIVES: To develop a biotherapy prepared from the infectious influenza A virus (A/Aichi/2/68 H3N2) and to verify its in vitro response. METHODS: The ultradiluted influenza virus solution was prepared in the homeopathic dilution 30dH, it was termed Influenzinum RC. The cellular alterations induced by this preparation were analyzed by optical and electron microscopy, MTT and neutral red assays. Glycolytic metabolism (PFK-1) was studied by spectrophotometric assay. Additionally, the production of tumor necrosis factor-α (TNF-α) by J774.G8 macrophage cells was quantified by ELISA before and after infection with H3N2 influenza virus and treatment. RESULTS: Influenzinum RC did not cause cytotoxic effects but induced morphological alterations in Madin-Darby canine kidney (MDCK) cells. After 30 days, a significant increase (p < 0.05) in mitosis rate was detected compared to control. MDCK mitochondrial activity was changed after treatment for 10 and 30 days. Treatment significantly diminished (p < 0.05) PFK-1 activity. TNF-α in biotherapy-stimulated J774.G8 macrophages indicated a significant (p < 0.05) increase in this cytokine when the cell supernatant was analyzed. CONCLUSION: Influenzinum RC altered cellular and biochemical features of MDCK and J774G8 cells.

Detection of avian paramyxoviruses in migratory and resident birds in the state of Rio de Janeiro, Brazil.

Paramyxoviruses and avian influenza viruses are present worldwide, and wild birds are known natural reservoirs of these viruses. This study monitored the circulation of these viruses in migratory and resident coastal birds captured in the state of Rio de Janeiro, Brazil. In total, 494 birds were trapped, and their fecal samples were collected and inoculated into embryonated chicken eggs. The allantoic fluids were evaluated using a hemagglutination test and PCR amplification of the genes of the M and L proteins of influenza A virus and paramyxovirus, respectively. Avian paramyxovirus was detected in 5 (1.01%) of the birds. The majority of these viruses were isolated from migratory birds classified into the order Charadriiformes (families Scolopacidae and Charadriidae). Four samples were characterized as avian paramyxovirus serotype-2 (APMV-2) by a hemagglutination inhibition test. These results reinforce the importance of continuous surveillance of wild species in Brazil.

Flavonoids in the therapy and prophylaxis of flu: a patent review.

INTRODUCTION: Influenza viruses are common agents of flu outbreaks, epidemics, and pandemics that have occurred through the centuries. Prevention and control of flu are of great clinical importance, since they cause serious damage to health, with a consequent impact on quality of life and economy of a country. Resistance against the current drugs justifies the development of new anti-influenza molecules. Flavonoids exhibit significant activity against flu through their anti-inflammatory and antiviral properties. The profile of these molecules makes them particularly promising as therapeutic agents against flu. AREAS COVERED: This review focus on the activity of flavonoids on different influenza virus targets as well as their use in patented pharmaceutical formulations. Twenty-one patents of these compounds for prophylaxis and treatment of influenza infection are discussed. EXPERT OPINION: The H1N1 influenza pandemic in 2009 resulted in a significant increase in the number of patents claiming pharmaceutical formulations for prophylaxis and treatment of flu. The research advances on flavonoids showing anti-influenza activity and the efforts made by researchers and industries consolidate the interest on new alternatives for the therapy of an infectious disease that represents a serious public health problem throughout the world.